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THE DECLINE OF A GIFTED AND SINCERE MAN

LIFETIME LYME EXPOSURES AND OTHER
TICK-BORNE INFECTIONS AND PRESIDENT BUSH

I recently was talking to a passionate Republican who was discussing the decreased ability of President Bush and his decreased memory, cognitive processing speed and speech fluency.

He had great respect for the President. He also felt very strongly something was going on that was missed.

Simply, the President has had behavior and cognition and emotional issues on and off for his entire life. He has had massive deer tick exposures. His huge EM bulls-eye rash was handled curiously and was assumed to be the only infection.

*****

Does Lyme Disease Explain Bush's Erratic Behavior?

Posted by Larkin
Published: Aug 10, 07 06:00 AM

The White House has revealed that President Bush received undisclosed treatment for Lyme Disease in August of 2006 as reported by ABC. Lyme Disease (Borreliosis) is a bacterial infection that is typically acquired by the bite of an infected tick. Wikipedia has the gory details of this ugly disease: The disease varies widely in its presentation, which may include a rash and flu-like symptoms in its initial stage, followed by the possibility of musculoskeletal, arthritic, neurologic, psychiatric and cardiac manifestations. In most cases of Lyme disease, symptoms can be eliminated with antibiotics, especially if treatment is begun early in the course of illness.

A percentage of patients with Lyme disease have symptoms that last months to years after treatment with antibiotics. These symptoms can include muscle and joint pains, arthritis, stiff neck, cognitive defects, neurological complaints or fatigue. The cause of these continuing symptoms is not yet known. There is some evidence that they may result from an autoimmune type of response, in which a person's immune system continues to respond even after the infection has been cleared, as well as evidence of ongoing infection with the spirochete.

The White House says that Bush was treated for "early, localized Lyme disease" after developing the characteristic bullseye rash. They provided a lame excuse for why Bush's disease was not disclosed earlier and then gave us the remarkable news that doctors have decided not to perform blood tests to determine the extent of his illness: White House spokesman Scott Stanzel said Bush's treatment was not disclosed earlier because it happened after his last physical, on Aug. 1, 2006. He said doctors decided not to perform blood tests for Lyme disease because the treatment worked for the one area where the president experienced a rash, and he never progressed to other symptoms or saw a recurrence.

I don't know about you but if I was in Bush's situation I certainly would have had the full battery of blood tests to determine the extent of my affliction with the disease. It certainly is bizarre that Bush's doctors would elect not to conduct such tests when there is clearly no downside to doing so. Why wouldn't they want to be absolutely certain about the extent of the disease? Especially, when the patient is the President of the United States?

The only answer I can come up with is that Bush's doctors already know how bad it is because he has had the disease for a number of years. The President's bizarre behavior during press conferences might be explained by the neurological and cognitive damage of the disease.

It certainly is disquieting that the White House thought it necessary to cover up the fact that Bush has been stricken with this serious disease. And for what reason? Actions like these will only create suspicion that the disease is worse than they are letting on.

wizbangblue.com/2007/08/10/does-lyme-disease-explain-bushs-erratic-behavior.php

Washington Post Quotes

A report of the president's recent medical examination said his case had "complete resolution" and was "without recurrence" since being treated last August. The illness, an infection carried by deer ticks that is prevalent in the Northeastern United States, had not been previously revealed.

While untreated Lyme disease can cause arthritis, an abnormal heart rhythm and problems with the nervous system, those complications usually can be prevented by taking antibiotics at an early stage of the infection. The medical record did not describe the details of the president's therapy.

Up to 15 percent of people treated for Lyme disease later complain of symptoms such as fatigue and muscle pain. Whether that is a consequence of the infection is uncertain and a matter of controversy. Chronic pain and tiredness are extremely common in adults; whether people who have had Lyme disease suffer from those problems in higher numbers is unknown.

"I wouldn't expect any problem at all for the president," said Gary Wormser, chief of infectious diseases at New York Medical College and an expert on Lyme disease. "He won't be impacted by this infection in the future."

Lyme disease, named after the town in Connecticut where the first cases were identified in the 1970s, causes a rash that is often its sole manifestation. Classically it is a large reddish oval with a lighter-colored center and is often described as looking like a target.

White House spokesman Scott Stanzel said Bush found a rash on the front of his lower left leg and alerted White House physicians.

While the Lyme organism Borrelia burgdorferi can sometimes be isolated in the skin or bloodstream — and antibodies to it can also eventually be detected in the blood — laboratory testing is often not done. That is because a person with a typical rash and a history of outdoor activity will be treated for the disease, regardless of what the tests show.

Without such tests, however, it is impossible to rule out a Lyme disease look-alike called STARI as the cause of the president's illness last summer. STARI stands for "Southern tick-associated rash illness." It also causes a target-like rash and is associated with a tick bite, but the causative organism has not been found.

STARI is common in Texas. The lone star tick is the species that transmits it. There are no documented cases of Lyme disease in the president's home state, where he spent much of last August on vacation.

[REALLY? THE VETS SURE REPORT DIFFERENT, AND MANY PATIENTS HAVE POSITIVE LYME TESTS, BABESIA TESTS AND EHRLICHIA TESTS AND BARTONELLA TESTS IN TEXAS.]

http://www.washingtonpost.com/wp-dyn/content/article/2007/08/08/AR2007080802268.html
[For actual knowledge of STARI read Dr. Masterson. Period].

****

Am. J. Trop. Med. Hyg., 44(5), 1991, pp. 469-474

Copyright © 1991 by The American Society of Tropical Medicine and Hygiene

Isolation of Borrelia burgdorferi from arthropods Collected in Texas

Glenna J. Teltow, Paul V. Fournier and Julie A. Rawlings

Microbiological Services Division, Bureau of Laboratories; and Zoonosis Control Division, Bureau of Veterinary Public Health, Texas Department of Health, Austin, Texass

The Texas Department of Health Laboratory cultured arthropods from November 1988 through December 1989 in an attempt to isolate Borrelia burgdorferi, the etiologic agent of Lyme disease. Spirochetes were isolated from eight of 1,093 pools of arthropods cultured. The spirochetal isolates were from several tick and one flea species, including Amblyomma americanum, A. maculatum, Ixodes scapularis, and Ctenocephalides felis. These 8 isolates reacted specifically when treated with monoclonal antibodies to B. burgdorferi. Polyacrylamide gel electrophoresis of six lysates showed them to be virtually identical with strain B31 of B. burgdorferi.

  • In Texas, there are 11 public health regions. Patients with Lyme disease reside in every public health region in Texas.
  • Borrelia burgdorferi, the agent of Lyme disease has been detected in Texas ticks.
  • Epidemiological evidence suggests Amblyomma americanum, the "Lone Star" tick, is the vector of Lyme disease in Texas. This is an aggressive species that will feed on a variety of hosts including humans. In a Texas Department of Health study conducted in 1990 and 1991, A. americanum ticks were gathered from nine Texas areas. Of the over 28,000 ticks collected, 26,901 or 95% were A. americanum. Visitors to any area with high vegetation are at considerable risk of being bitten by lone star ticks and are at risk of acquiring Lyme disease.
  • There are four reportable tick-borne illnesses in Texas: ehrlichiosis, Lyme disease, rocky mountain spotted fever and tick-borne relapsing fever. Patients in Texas have also been diagnosed with babesiosis, a malaria-like, tick-borne illness with recurring fevers.
  • Failure to report is a Class B misdemeanor under the Texas Health and Safety Code, Section 81.049, but this provision is rarely, if ever, enforced.
  • Texas is a passive surveillance state; and it is likely that there is considerable underreporting of tick-borne illnesses.

www.txlda.org/facts.htm

*****

Journal of Wildlife Diseases, 25(1), 1989, pp. 47-51
© Wildlife Disease Association 1989

Borrelia sp. infection in coyotes, black-tailed jack rabbits and desert cottontails in southern Texas

EC Burgess and LA Windberg

ABSTRACT

Coyotes (Canis latrans) from southern Texas were sampled for antibodies to Borrelia burgdorferi from 1980 to 1986; black-tailed jack rabbits (Lepus californicus) and desert cottontails (Sylvilagus audubonii) were sampled in 1986. Coyote fetuses, adult coyote kidneys, and black-tailed jack rabbit and desert cottontail kidneys were cultured for B. burgdorferi in 1986. Results of indirect immunofluorescent antibody (IFA) tests for B. burgdorferi in coyotes were as follows (number positive at a dilution of greater than or equal to 1:128/number tested): 1980 (0 of 30), 1981 (0 of 21), 1982 (0 of 53), 1983 (0 of 78), 1984 (47 of 97), 1985 (20 of 88), and 1986 (42 of 80). Eight of 26 black-tailed jack rabbits and two of seven desert cottontails tested in 1986 had IFA titers to B. burgdorferi of greater than or equal to 1:128. Borrelia burgdorferi was isolated from one of five coyote fetuses, three of 31 adult coyote kidneys, and two of 10 black-tailed jack rabbit kidneys in 1986. These results indicate that B. burgdorferi infection has been present in coyotes in Texas, at least since 1984 and that transplacental transmission occurs.

***

Results: During 1992?1998, a total of 88,967 cases of Lyme disease was reported to CDC by 49 states and the District of Columbia.

www.cdc.gov/mmwr/PDF/ss/ss4903.pdf


Sample Lyme and Other Tick Infection Work
Completed in Texas Institutions
or Reporting Infection in TX

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sodA is essential for virulence of Borrelia burgdorferi in the murine model of Lyme disease.

Esteve-Gassent MD, Elliott NL, Seshu J.

South Texas Center for Emerging Infectious Diseases and Department of Biology, The University of Texas at San Antonio, San Antonio, TX-78249.

Abstract Borrelia burgdorferi, the causative agent of Lyme disease, has a limited set of genes to combat oxidative/nitrosative stress encountered in its tick vector or mammalian hosts. We inactivated the gene encoding for superoxide dismutase A (sodA, bb0153), an enzyme mediating the dismutation of superoxide anions and examined the in vitro and in vivo phenotype of the mutant. There were no significant differences in the in vitro growth characteristics of the sodA mutant compared to the control strains. Microscopic analysis of viability of spirochetes revealed greater percentage of cell death upon treatment of sodA mutant with superoxide generators compared to its controls. Infectivity analysis in C3H/HeN mice following intradermal needle inoculation of 10(3) or 10(5) spirochetes per mouse revealed complete attenuation of infectivity for the sodA mutant compared to control strains at 21 days post-infection. The sodA mutant was more susceptible to the effects of activated macrophages and neutrophils suggesting that its in vivo phenotype is partly due to the killing effects of activated immune cells. These studies indicate that SodA plays an important role in combating oxidative stress and is essential for the colonization and dissemination of B. burgdorferi in the murine model of Lyme disease.

PMID: 19040638 [PubMed - as supplied by publisher]

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Borrelia burgdorferi lacking DbpBA exhibits an early survival defect during experimental infection.

Weening EH, Parveen N, Trzeciakowski JP, Leong JM, Höök M, Skare JT.

Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.

Several Borrelia burgdorferi genes induced under mammalian host conditions have been purported to be important in Lyme disease pathogenesis based on their binding to host structures. These genes include the dbpBA locus, whose products bind host decorin and glycosoaminoglycans. Recently, the dbpBA genes were reported to be involved in borrelial infectivity. Here we extended the previous observations by using culture and quantitative PCR to evaluate low- and high-dose murine infection by a Delta dbpBA::Gent(r) derivative of B. burgdorferi strain B31. The results indicate that the Delta dbpBA::Gent(r) mutant is attenuated in the ability to initially colonize and then persist in multiple tissues. The mutant exhibited a colonization defect as early as 3 days postinfection, before the development of an adaptive immune response, and after low-dose infection of SCID mice, which are deficient in adaptive immunity. These findings suggest that the inability to adhere to host decorin may promote clearance of B. burgdorferi, presumably via innate immune mechanisms. In a high-dose infection, the mutant disseminated to several tissues, particularly joint tissue, but it was generally cleared from these tissues by 3 weeks postinfection. Finally, following high-dose infection of SCID mice, the dbpBA mutant exhibited only a mild colonization defect, suggesting that the adaptive response is involved in the clearance of the mutant in immunocompetent mice. Taken together, these results suggest that the DbpBA proteins facilitate the colonization of multiple tissues by B. burgdorferi and are required for optimal resistance to both innate and adaptive immune mechanisms following needle inoculation.

Publication Types:
PMID: 18809667 [PubMed - in process]

PMCID: PMC2583571 [Available on 2009/06/01]


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Deletion of BBA64, BBA65, and BBA66 loci does not alter the infectivity of Borrelia burgdorferi in the murine model of Lyme disease.

Maruskova M, Seshu J.

South Texas Center for Emerging Infectious Diseases and Department of Biology, The University of Texas at San Antonio, San Antonio, Texas 78249, USA.

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its tick vector versus vertebrate hosts. Whole-genome transcriptional profile analysis of B. burgdorferi, propagated in vitro under mammalian-host-specific conditions, revealed significant upregulation of several linear plasmid 54 (lp54)-encoded open reading frames (ORFs). Among these ORFs, BBA64, BBA65, and BBA66 have been shown to be upregulated in response to multiple mammalian-host-specific signals. Recently, we determined that there was no significant difference in the ability of BBA64(-) mutant to infect C3H/HeN mice compared to its isogenic control strains, suggesting that B. burgdorferi might utilize multiple, functionally related determinants to establish infection. We further generated BBA65(-) and BBA66(-) single mutants in a noninfectious, lp25(-) clonal isolate of B. burgdorferi strain B31 (ML23) and complemented them with the minimal region of lp25 (BBE22) required for restoring the infectivity. In addition, we generated a BBA64(-) BBA65(-) BBA66(-) triple mutant using an infectious, clonal isolate of B. burgdorferi strain B31 (5A11) that has all of the infection-associated plasmids. There were no significant differences in the ability to isolate viable spirochetes from different tissues of C3H/HeN mice infected via intradermal needle inoculation with either the individual single mutants or the triple mutant compared to their respective isogenic parental strains at days 21 and 62 postinfection. These observations suggest that B. burgdorferi can establish infection in the absence of expression of BBA64, BBA65, and BBA66 in the murine model of Lyme disease.

Publication Types:
PMID: 18765733 [PubMed - indexed for MEDLINE]

PMCID: PMC2573326 [Available on 2009/03/01]


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Transcriptional interplay among the regulators Rrp2, RpoN and RpoS in Borrelia burgdorferi.

Ouyang Z, Blevins JS, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

The RpoN-RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN-RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.

Publication Types:
PMID: 18757798 [PubMed - indexed for MEDLINE]

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Assessment of decorin-binding protein A to the infectivity of Borrelia burgdorferi in the murine models of needle and tick infection.

Blevins JS, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. jon.blevins@utsouthwestern.edu

BACKGROUND: Decorin-binding proteins (Dbps) A and B of Borrelia burgdorferi, the agent of Lyme disease, are surface-exposed lipoproteins that presumably bind to the extracellular matrix proteoglycan, decorin. B. burgdorferi infects various tissues including the bladder, heart, joints, skin and the central nervous system, and the ability of B. burgdorferi to bind decorin has been hypothesized to be important for this disseminatory pathogenic strategy. RESULTS: To determine the role of DbpBA in the infectious lifecycle of B. burgdorferi, we created a DbpBA-deficient mutant of B. burgdorferi strain 297 and compared the infectious phenotype of the mutant to the wild-type strain in the experimental murine model of Lyme borreliosis. The mutant strain exhibited a 4-log decrease in infectivity, relative to the wild-type strain, when needle inoculated into mice. Upon complementation of the DbpBA-mutant strain with DbpA, the wild-type level of infectivity was restored. In addition, we demonstrated that the DbpBA-deficient mutant was able to colonize Ixodes scapularis larval ticks after feeding on infected mice and persist within the ticks during the molt to the nymphal state. Moreover, surprisingly, the DbpBA-mutant strain was capable of being transmitted to naïve mice via tick bite, giving rise to infected mice. CONCLUSION: These results suggest that DbpBA is not required for the natural tick-transmission process to mammals, despite inferences from needle-inoculation experiments implying a requirement for DbpBA during mammalian infection. The combined findings also send a cautionary note regarding how results from needle-inoculation experiments with mice should be interpreted.

Publication Types:
PMID: 18507835 [PubMed - indexed for MEDLINE]

PMCID: PMC2430964


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Ultrastructural evidence of the ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with Rickettsia, Borrelia, and a flavivirus.

Popov VL, Korenberg EI, Nefedova VV, Han VC, Wen JW, Kovalevskii YV, Gorelova NB, Walker DH.

Department of Pathology, The University of Texas Medical Branch, Center for Biodefense and Emerging Infectious Diseases, Galveston, Texas 77555, USA.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

Publication Types:
PMID: 18171109 [PubMed - indexed for MEDLINE]

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Role of the BBA64 locus of Borrelia burgdorferi in early stages of infectivity in a murine model of Lyme disease.

Maruskova M, Esteve-Gassent MD, Sexton VL, Seshu J.

South Texas Center for Emerging Infectious Diseases and Department of Biology, The University of Texas at San Antonio, San Antonio, Texas 7824, USA.

Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.

Publication Types:
PMID: 17984202 [PubMed - indexed for MEDLINE]

PMCID: PMC2223643


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Spirochetemia caused by Borrelia turicatae infection in 3 dogs in Texas.

Whitney MS, Schwan TG, Sultemeier KB, McDonald PS, Brillhart MN.

Texas Veterinary Medical Diagnostic Laboratory, Texas A&M University, Amarillo, TX, USA. whitneym@missouri.edu

Spirochetemia was diagnosed in 2 Siberian Huskies and a Rottweiler from the northwestern region of Texas between June 1999 and October 2001. Clinical findings were nonspecific; tick exposure was documented in 2 of the dogs. Hematologic abnormalities included anemia (n=2), neutrophilia (n=2, including 1 with a left shift), lymphopenia (n=3), eosinopenia (n=3), and thrombocytopenia (n=2). One anemic dog had a positive Coombs' test. In 1 dog, Western blot analysis of serum yielded multiple positive bands with B turicatae lysate, indicating the spirochetemia most likely was due to B turicatae infection. In 2 dogs, spirochetes were cultured from the blood and identified using DNA analysis as Borrelia turicatae; 1 of these dogs also was seropositive for Ehrlichia canis and B burgdorferi. In 2 cases, spirochetemia was more prominent in blood smears prepared immediately after sample collection than in smears prepared from EDTA blood. Two dogs recovered with doxycycline treatment; 1 dog declined clinically despite treatment and was euthanized. B turicatae is the agent of tick-borne (endemic) relapsing fever in humans and is distinct from B burgdorferi, the agent of Lyme disease; however, serologic cross-reactivity may occur. B turicatae is transmitted by the soft tick, Ornithodoros turicata, and infection should be considered in dogs with spirochetemia and possible exposure to the tick vector.

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PMID: 17523100 [PubMed - indexed for MEDLINE]

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Split target specificity of ResT: a design for protein delivery, site selectivity and regulation of enzyme activity?

Jayaram M.

Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA. jayaram@icmb.utexas.edu

The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.

Publication Types:
PMID: 17462008 [PubMed - indexed for MEDLINE]

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Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi.

Blevins JS, Revel AT, Smith AH, Bachlani GN, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9048, USA.

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.

Publication Types:
PMID: 17220265 [PubMed - indexed for MEDLINE]

PMCID: PMC1828772


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Evidence that RpoS (sigmaS) in Borrelia burgdorferi is controlled directly by RpoN (sigma54/sigmaN).

Smith AH, Blevins JS, Bachlani GN, Yang XF, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.

The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.

Publication Types:
PMID: 17158681 [PubMed - indexed for MEDLINE]

PMCID: PMC1855718


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Borrelia burgdorferi alters its gene expression and antigenic profile in response to CO2 levels.

Hyde JA, Trzeciakowski JP, Skare JT.

Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.

The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO(2), serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO(2), 3% H(2), 0.087 ppm O(2)) and microaerophilic (1% CO(2), 3.48 ppm O(2)) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO(2). In this study we identified CO(2) as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO(2), independent of O(2) levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO(2). Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO(2), indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO(2) may be an additional cue for borrelial host adaptation and gene regulation.

Publication Types:
PMID: 17098904 [PubMed - indexed for MEDLINE]

PMCID: PMC1797391


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Identification of potential virulence determinants by Himar1 transposition of infectious Borrelia burgdorferi B31.

Botkin DJ, Abbott AN, Stewart PE, Rosa PA, Kawabata H, Watanabe H, Norris SJ.

Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77225-0708, USA.

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

Publication Types:
PMID: 17015459 [PubMed - indexed for MEDLINE]

PMCID: PMC1698074


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Transcriptional profiling of Borrelia burgdorferi containing a unique bosR allele identifies a putative oxidative stress regulon.

Hyde JA, Seshu J, Skare JT.

Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Science Center, College Station, 77843-1114, USA.

Borrelia burgdorferi regulates gene expression in response to environmental conditions, including temperature, pH, redox potential and host factors. B. burgdorferi encodes a PerR homologue designated BosR, which presumably serves as a global regulator of genes involved in the oxidative stress response. Infectious B. burgdorferi strain B31 is resistant to oxidative stressors in vitro, whereas the non-infectious isolate was sensitive due, in part, to a point mutation that converts an arginine to a lysine at residue 39 of BosR. Subsequent insertional inactivation of this bosRR39K allele (bosRR39K : : kan(R)) restored resistance to oxidative stressors. These observations suggest that the B. burgdorferi non-infectious bosRR39K : : kan(R) strain may transcribe genes that are also expressed in infectious B. burgdorferi cells, but are repressed in the bosRR39K background, thus explaining the different oxidative stress phenotypes observed between these isolates. To test this hypothesis, macroarray technology and quantitative RT-PCR were utilized to compare the transcriptional profiles from the isogenic bosRR39K and bosRR39K : : kan(R) isolates. Array data indicated that 88 ORFs were significantly expressed in the absence of BosRR39K. Since most affected genes mapped to the chromosome, it is likely that these genes define an important physiologic response for B. burgdorferi. Included within the genes identified was the detoxification gene sodA, as well as other loci not overtly linked to oxidative stress. These results suggest that a putative BosR regulon, as defined by the bosRR39K allele, is required to combat toxic oxidative intermediates, but may also be involved in adaptive strategies that are independent of reactive oxygen species.

Publication Types:
PMID: 16946255 [PubMed - indexed for MEDLINE]

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Differential effects of alcohols on conformational switchovers in alpha-helical and beta-sheet protein models.

Perham M, Liao J, Wittung-Stafshede P.

Department of Chemistry, Keck Center for Structural Computational Biology, Rice University, 6100 Main Street, Houston, Texas 77251, USA.

Organic solvents may induce non-native structures of proteins that mimic folding intermediates and/or conformations that occur in proximity to biological membranes. Here we systematically investigate the effects of simple (i.e., MeOH and EtOH) and fluorinated (i.e., trifluoroethanol, TFE) alcohols on the secondary structure and thermodynamic stability of two complementary model proteins using a combination of circular dichroism, fluorescence, and Fourier transform infrared (FTIR) detection methods. The selected proteins are alpha-helical Borrelia burgdorferi VlsE and beta-sheet human mitochondrial co-chaperonin protein 10 (cpn10). We find that switches between VlsE's native and non-native superhelical and beta-sheet structures readily occur (pH 7, 20 degrees C). The pathway depends on the alcohol: addition of MeOH induces a transition to a superhelical structure that is followed by conversion to beta-structure, whereas EtOH only unfolds the protein. TFE unfolds VlsE at low percentages but promotes the formation of a superhelical state upon further additions. For cpn10, both MeOH and TFE additions govern initial unfolding; however, further additions of MeOH result in the formation of a non-native beta-structure, whereas subsequent additions of TFE induce a superhelical structure. EtOH additions promptly unfold and precipitate cpn10. Both VlsE's and cpn10's non-native structures exhibit high stability toward chemical and thermal perturbations. This study demonstrates that in response to different alcohols, polypeptides can readily adopt both alpha- and beta-enriched conformations. The biological significance of these findings is discussed.

Publication Types:
PMID: 16784225 [PubMed - indexed for MEDLINE]

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Detection of Babesia and Anaplasma species in rabbits from Texas and Georgia, USA.

Yabsley MJ, Romines J, Nettles VF.

Southeastern Cooperative Wildlife Disease Study and Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA. myabsley@uga.edu

Rabbits have been shown to harbor a suite of zoonotic organisms, including a Babesia species, Borrelia burgdorferi, and Anaplasma phagocytophilum. In this study, we conducted a molecular survey for various tick-borne pathogens in three species of rabbits from Texas and Georgia. Of 18 black-tailed jackrabbits (Lepus californicus) tested from Texas, six (28%) were polymerase chain reaction (PCR) positive for Babesia, and nucleotide sequencing revealed two distinct species or strains. Two jackrabbits were infected with a Babesia species or strain (Babesia sp. A) that was nearly identical (99.9%) to a piroplasm previously detected in humans from Washington state, and the remaining four jackrabbits were infected with a Babesia species (Babesia sp. B) that was most similar (99.7%) to a Babesia species detected in cottontail rabbits from Massachusetts and humans from Kentucky and Missouri. Eleven (61%) black-tailed jackrabbits were positive for A. bovis, and one was positive for A. phagocytophilum. Two of four desert cottontails (Sylvilagus audubonii) from Texas were positive for the Babesia sp. B, and one desert cottontail each was positive for A. bovis and A. phagocytophilum. One of these desert cottontails was coinfected with the Babesia sp. B and A. phagocytophilum, and five jackrabbits were coinfected with Babesia species and A. bovis. Of 19 eastern cottontails (S. floridanus) from Georgia, only one (5.3%) was positive for A. phagocytophilum, and three (15.8%) were positive for A. bovis. No rabbits from Texas or Georgia were positive for Borrelia species. The only tick species detected on the Texas and Georgia rabbits was the rabbit tick, Haemaphysalis leporispalustris. These data extend the geographic and host range of these pathogens, and because both the Babesia species and A. phagocytophilum are potential zoonotic pathogens, it is important to be aware that these organisms are enzootic in parts of the southern United States.

Publication Types:
PMID: 16584322 [PubMed - indexed for MEDLINE]

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The dynamic proteome of Lyme disease Borrelia.

Norris SJ.

Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, TX 77225-0708, USA. Steven.J.Norris@uth.tmc.edu

The proteome of the spirochete bacterium Borrelia burgdorferi, the tick-borne agent of Lyme disease, has been characterized by two different approaches using mass spectrometry, providing a launching point for future studies on the dramatic changes in protein expression that occur during transmission of the bacterium between ticks and mammals.

Publication Types:
PMID: 16563176 [PubMed - indexed for MEDLINE]

PMCID: PMC1557748


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The mystery of Morgellons disease: infection or delusion?

Savely VR, Leitao MM, Stricker RB.

South Austin Family Practice Clinic, Austin, Texas, USA.

Morgellons disease is a mysterious skin disorder that was first described more than 300 years ago. The disease is characterized by fiber-like strands extruding from the skin in conjunction with various dermatologic and neuropsychiatric symptoms. In this respect, Morgellons disease resembles and may be confused with delusional parasitosis. The association with Lyme disease and the apparent response to antibacterial therapy suggest that Morgellons disease may be linked to an undefined infectious process. Further clinical and molecular research is needed to unlock the mystery of Morgellons disease.

Publication Types:
PMID: 16489838 [PubMed - indexed for MEDLINE]

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Inactivation of the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi.

Seshu J, Esteve-Gassent MD, Labandeira-Rey M, Kim JH, Trzeciakowski JP, Höök M, Skare JT.

Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Science Center, 407 Reynolds Medical Building, College Station, 77843, USA.

Borrelia burgdorferi, the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surface-exposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi. Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.

Publication Types:
PMID: 16468997 [PubMed - indexed for MEDLINE]

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Neuralgia and demyelinating plaques: MS or lyme disease?

Savely G.

South Austin Family Practice Clinic, Austin, Texas, USA.

Publication Types:
PMID: 16152811 [PubMed - indexed for MEDLINE]

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Primary cutaneous B-cell lymphoma.

Bogle MA, Riddle CC, Triana EM, Jones D, Duvic M.

Department of Dermatology, University of Texas and M. D. Anderson Cancer Center, Houston, Texas 77030, USA. mabogle@hotmail.com

Primary cutaneous B-cell lymphomas include extranodal marginal zone B-cell lymphoma, follicular lymphoma, large B-cell lymphoma, and, rarely, mantle cell lymphoma. Our purpose in conducting this review was to determine the clinical and behavioral characteristics of primary cutaneous B-cell lymphomas, their relationship to infectious triggers, and therapeutic response. We conducted a retrospective chart review of 23 adult patients presenting to the dermatology clinic at M. D. Anderson Cancer Center with primary cutaneous B-cell lymphoma between January 1999 and May 2003. Primary cutaneous B-cell lymphomas generally present on the head and neck, with the trunk and extremities afflicted to a lesser extent. Patients were found to have serologic evidence of prior infection with Borrelia burgdorferi (n = 10), Helicobacter pylori (n = 5), and Epstein-Barr virus (n = 6). Overall, treatment of primary cutaneous B-cell lymphoma should involve multiple modalities; however, specific treatment aimed at concurrent or suspected infection, particularly B burgdorferi, is a helpful adjunct and may achieve complete remission in a small subset of patients.

PMID: 16112357 [PubMed - indexed for MEDLINE]

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Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

Yang XF, Lybecker MC, Pal U, Alani SM, Blevins J, Revel AT, Samuels DS, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, 75390-9048, USA.

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.

Publication Types:
PMID: 15995197 [PubMed - indexed for MEDLINE]

PMCID: PMC1169512


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Multicomponent Lyme vaccine: three is not a crowd.

Brown EL, Kim JH, Reisenbichler ES, Höök M.

The Center for Extracellular Matrix Biology, Texas A&M University System Health Science Center, Albert B. Alkek Institute of Biosciences and Technology, 2121 W, Holcombe Blvd., Suite 603, Houston, TX 77030, USA. ebrown2@uh.edu

Lyme disease is caused by the spirochete Borrelia burgdorferi and it is the most common vector-borne disease in the United States. Disseminated spirochetes can persist in various tissues and can result in a variety of different disease manifestations. Vaccination trials testing various lipoprotein candidates have yielded mixed results despite the generation of robust antibody titers. Data presented in this report demonstrate that a combination vaccine composed of DbpA, BBK32 and OspC is more effective than single or double component formulations and that the ratio of each component dramatically impacts vaccine efficacy when tested in protection experiments against Borrelia following needle inoculation.

Publication Types:
PMID: 15882529 [PubMed - indexed for MEDLINE]

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bptA (bbe16) is essential for the persistence of the Lyme disease spirochete, Borrelia burgdorferi, in its natural tick vector.

Revel AT, Blevins JS, Almazán C, Neil L, Kocan KM, de la Fuente J, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Borrelia burgdorferi (Bb), the agent of Lyme disease, is a zoonotic spirochetal bacterium that depends on arthropod (Ixodes ticks) and mammalian (rodent) hosts for its persistence in nature. The quest to identify borrelial genes responsible for Bb's parasitic dependence on these two diverse hosts has been hampered by limitations in the ability to genetically manipulate virulent strains of Bb. Despite this constraint, we report herein the inactivation and genetic complementation of a linear plasmid-25-encoded gene (bbe16) to assess its role in the virulence, pathogenesis, and survival of Bb during its natural life cycle. bbe16 was found to potentiate the virulence of Bb in the murine model of Lyme borreliosis and was essential for the persistence of Bb in Ixodes scapularis ticks. As such, we have renamed bbe16 a gene encoding borrelial persistence in ticks (bpt)A. Although protease accessibility experiments suggested that BptA as a putative lipoprotein is surface-exposed on the outer membrane of Bb, the molecular mechanism(s) by which BptA promotes Bb persistence within its tick vector remains to be elucidated. BptA also was shown to be highly conserved (>88% similarity and >74% identity at the deduced amino acid levels) in all Bb sensu lato strains tested, suggesting that BptA may be widely used by Lyme borreliosis spirochetes for persistence in nature. Given Bb's absolute dependence on and intimate association with its arthropod and mammalian hosts, BptA should be considered a virulence factor critical for Bb's overall parasitic strategy.

Publication Types:
PMID: 15860579 [PubMed - indexed for MEDLINE]

PMCID: PMC1100799


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Immune evasion of the Lyme disease spirochetes.

Bubeck-Martinez S.

Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. martinezsb@uthscsa.edu <martinezsb@uthscsa.edu>

The Lyme disease spirochetes, Borrelia burgdorferi sensu lato, have adapted very well to both surviving and persisting in the mammalian host despite a strong host antibody response. It appears that both temporal and spatial regulation of outer surface proteins have contributed to this persistence. The spirochetes are able to bind fH and FHL-1 to their surface, resulting in decreased complement activation. In addition, the organisms have taken advantage of components of tick saliva to aid in their initial immune evasion and dissemination. Studies leading to these conclusions are reviewed here.

Publication Types:
PMID: 15569625 [PubMed - indexed for MEDLINE]

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A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi.

Seshu J, Boylan JA, Hyde JA, Swingle KL, Gherardini FC, Skare JT.

Department of Medical Microbiology and Immunology, 407 Reynolds Medical Building, Texas A&M University Health Science Center, College Station, TX 77843, USA.

Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi. When we tested the sensitivity of B. burgdorferi to ROS, one clonal non-infectious B. burgdorferi isolate exhibited hypersensitivity to t-butyl hydroperoxide when compared with infectious B. burgdorferi and other non-infectious isolates. Sequence analysis indicated that the hypersensitive non-infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non-infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t-butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter-lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA). In addition, we determined that cells lacking BosRR39K synthesized fourfold greater levels of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.

Publication Types:
PMID: 15554974 [PubMed - indexed for MEDLINE]

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Effects of vlsE complementation on the infectivity of Borrelia burgdorferi lacking the linear plasmid lp28-1.

Lawrenz MB, Wooten RM, Norris SJ.

Department of Pathology and Laboratory Medicine, Graduate School of Biomedical Sciences, University of Texas--Houston Health Science Center, USA.

The loss of linear plasmid lp28-1, which contains the vls antigenic variation locus, is associated with reduced infectivity of Borrelia burgdorferi in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable B. burgdorferi lacking lp25 and lp56, was used to determine the effect of trans expression of vlsE on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and vlsE. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured B. burgdorferi, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact vls antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the vlsE shuttle plasmid and was examined for vlsE sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion. Lack of vlsE sequence variation on the shuttle plasmid thus did not result in clearance of the trans-complemented strain in immunocompetent mice under the conditions tested.

Publication Types:
PMID: 15501789 [PubMed - indexed for MEDLINE]

PMCID: PMC523020


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BBK32, a fibronectin binding MSCRAMM from Borrelia burgdorferi, contains a disordered region that undergoes a conformational change on ligand binding.

Kim JH, Singvall J, Schwarz-Linek U, Johnson BJ, Potts JR, Höök M.

Center for Extracellular Matrix Biology, Albert B. Alkek Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Houston, Texas 77030, USA.

BBK32 is a fibronectin-binding lipoprotein on Borrelia burgdorferi, the causative agent of Lyme disease. Analysis using secondary structure prediction programs suggested that BBK32 is composed of two domains, an N-terminal segment lacking well defined secondary structure and a C-terminal segment composed largely of alpha-helices. Analysis of purified recombinant forms of the two domains by circular dichroism spectroscopy, gel permeation chromatography, and intrinsic viscosity determination were consistent with an N-terminal-extended, unstructured segment and a C-terminal globular domain in BBK32. Solid phase binding experiments suggest that the unstructured N-terminal domain binds fibronectin. Analysis of changes in circular dichroism spectra of the N-terminal segment of BBK32 upon binding of the N-terminal domain of fibronectin revealed an increase in beta-sheet content in the complex. Hence, BBK32, which belongs to a different family of proteins and shows no overall sequence similarity with the fibronectin binding MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) of Gram-positive bacteria, binds fibronectin by a mechanism that is reminiscent of the "tandem beta-zipper" previously demonstrated for the fibronectin binding of streptococcal adhesins.

Publication Types:
PMID: 15292204 [PubMed - indexed for MEDLINE]

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The luxS gene is not required for Borrelia burgdorferi tick colonization, transmission to a mammalian host, or induction of disease.

Blevins JS, Revel AT, Caimano MJ, Yang XF, Richardson JA, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, 75390, USA.

luxS mutants of Borrelia burgdorferi strain 297 naturally colonized their arthropod (Ixodes scapularis) vector, were maintained in ticks throughout the molting process (larvae to nymphs), were tick transmitted to uninfected mice, and elicited histopathology in mice indistinguishable from that induced by wild-type B. burgdorferi.

Publication Types:
PMID: 15271949 [PubMed - indexed for MEDLINE]

PMCID: PMC470628


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Erratum in:
  • Infect Immun. 2004 Apr;72(4):2456.

Dissolved oxygen levels alter gene expression and antigen profiles in Borrelia burgdorferi.

Seshu J, Boylan JA, Gherardini FC, Skare JT.

Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.

The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in B. burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B. burgdorferi was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in B. burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

Publication Types:
PMID: 14977964 [PubMed - indexed for MEDLINE]

PMCID: PMC356058


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The response regulator Rrp2 is essential for the expression of major membrane lipoproteins in Borrelia burgdorferi.

Yang XF, Alani SM, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.

Borrelia burgdorferi (Bb), the agent of Lyme disease, exists in nature through a complex enzootic life cycle that involves both ticks and mammals. As Bb transitions between its two diverse niches, profound adaptive changes occur that are reflected in differential patterns of gene expression, particularly involving lipoprotein genes. Using a mutagenesis approach, we show that Rrp2 (gene BB0763), one of the proteins predicted by the Bb genome (www.tigr.org) to be a response regulator of a two-component sensory transduction system, is a pivotal regulator governing the expression of major membrane lipoproteins such as OspC, DbpA, and Mlp8, as well as many other mammalian infection-associated immunogens of Bb. Sequence analysis additionally suggested that Rrp2 is a bacterial enhancer-binding protein, essential for sigma54-dependent gene activation. Mutagenesis of a key amino acid residue within a putative activation domain revealed that Rrp2 controlled lipoprotein expression by governing the expression of the alternative sigma-factor sigmas in a sigma54-dependent manner. We therefore propose a signal transduction pathway involving Rrp2, sigma54, and sigmas, which in concert control the expression of key lipoproteins and other infection-associated immunogens in Bb.

Publication Types:
PMID: 12949258 [PubMed - indexed for MEDLINE]

PMCID: PMC196916


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Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi.

Yang XF, Hübner A, Popova TG, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

The Mlp (multicopy lipoproteins) family is one of many paralogous protein families in Borrelia burgdorferi. To examine the extent to which the 10 members of the Mlp family in B. burgdorferi strain 297 might be differentially regulated, antibodies specific for each of the Mlps were developed and used to analyze the protein expression profiles of individual Mlps when B. burgdorferi replicated under various cultivation conditions. All of the Mlps were upregulated coordinately when B. burgdorferi was cultivated at either elevated temperature, reduced culture pH, or increased spirochete cell density. Inasmuch as the expression of OspC is influenced by a novel RpoN-RpoS regulatory pathway, similar induction patterns for OspC and the Mlp paralogs prompted an assessment of whether the RpoN-RpoS pathway also was involved in Mlp expression. In contrast to wild-type B. burgdorferi, both RpoN- and RpoS-deficient mutants were unable to upregulate OspC or the Mlp paralogs when grown at lower pH (6.8), indicating that pH-mediated regulation of OspC and Mlp paralogs is dependent on the RpoN-RpoS pathway. However, when RpoN- or RpoS-deficient mutants were shifted from 23 degrees C to 37 degrees C or were cultivated to higher spirochete densities, some induction of the Mlps still occurred, whereas OspC expression was abolished. The combined findings suggest that the Mlp paralogs are coordinately regulated as a family and have an expression profile similar, but not identical, to that of OspC. Although Mlp expression as a family is influenced by the RpoN-RpoS regulatory pathway, there exists at least one additional layer of gene regulation, yet to be elucidated, contributing to Mlp expression in B. burgdorferi.

Publication Types:
PMID: 12933844 [PubMed - indexed for MEDLINE]

PMCID: PMC187337


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The absence of linear plasmid 25 or 28-1 of Borrelia burgdorferi dramatically alters the kinetics of experimental infection via distinct mechanisms.

Labandeira-Rey M, Seshu J, Skare JT.

Department of Medical Microbiology and Immunology, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.

The 25-kb linear plasmid lp25 and one of the 28-kb linear plasmids (lp28-1) are required for experimental infection in Borrelia burgdorferi, the etiologic agent of Lyme disease. The loss of these plasmids either eliminates infectivity (lp25) or significantly increases the 50% infective dose during a 2-week infection period (lp28-1). This study assessed the kinetics of bacterial dissemination in C3H/HeN mice infected with B. burgdorferi lacking either lp25 or lp28-1, as well as their wild-type parent, and tracked the development of specific borrelial antibodies over a 3-week period. The results indicated that the wild type and the lp28-1(-) strains were able to disseminate throughout the host, whereas the lp25(-) strain was cleared within 48 h of inoculation. While the wild-type B. burgdorferi persisted in tissues for the duration of the study, the lp28-1(-) mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1(-) mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1(-) mutant was able to disseminate and persist in C3H-scid mice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis.

Publication Types:
PMID: 12874340 [PubMed - indexed for MEDLINE]

PMCID: PMC166013


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Effect of complement component C3 deficiency on experimental Lyme borreliosis in mice.

Lawrenz MB, Wooten RM, Zachary JF, Drouin SM, Weis JJ, Wetsel RA, Norris SJ.

Program in Microbiology and Molecular Genetics and Department of Pathology and Laboratory Medicine, Graduate School of Biomedical Sciences, University of Texas-Houston Health Science Center, Houston, Texas, USA.

Mice deficient in complement component C3 (C3(-/-)) and syngeneic C57BL/6 control mice were challenged with Borrelia burgdorferi to determine the role of complement in immune clearance and joint histopathology during experimental Lyme borreliosis. Tibiotarsal joint, ear, and heart tissues were monitored for spirochete numbers at 2, 4, 8, and 12 weeks postinoculation with 10(5) B. burgdorferi B31 clone 5A4 by using quantitative real-time PCR. The spirochete load in joint and ear tissue remained higher in the C3(-/-) mice than in the wild-type counterparts throughout the 12-week study, whereas the numbers in heart tissue of both groups of mice decreased substantially at 8 to 12 weeks postinfection. Histopathology scores for joint tissue were generally higher in the C3(-/-) mice compared to C57BL/6 controls at 2 and 4 weeks postinfection, which may reflect the presence of higher numbers of bacteria in the joints at these early time points. Levels of anti-B. burgdorferi immunoglobulin G tended to be reduced in the C3(-/-) mice compared to control mice. Furthermore, a 5.5-fold-lower number of the complement-sensitive Borrelia garinii was needed to infect C3(-/-) mice compared to C57BL/6 mice, indicating that its sensitivity to complement is one barrier to infection of the mouse model by B. garinii. These results indicate that the complement system may be important in controlling the early dissemination and progression of B. burgdorferi infection.

Publication Types:
PMID: 12874322 [PubMed - indexed for MEDLINE]

PMCID: PMC165993


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Decorin-binding sites in the adhesin DbpA from Borrelia burgdorferi: a synthetic peptide approach.

Pikas DS, Brown EL, Gurusiddappa S, Lee LY, Xu Y, Höök M.

Center for Extracellular Matrix Biology, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA.

Lyme disease is caused by the spirochete Borrelia burgdorferi following transmission from infected Ixodes ticks to human hosts. Following colonization of the skin, spirochetes can disseminate throughout the body, resulting in complications that can include ocular, cardiac, neural, and skeletal disease. We have previously shown that B. burgdorferi expresses two closely related decorin-binding adhesins (DbpA and DbpB) of the MSCRAMM (microbial surface component recognizing adhesive matrix molecule) type that can mediate bacterial attachment to extracellular matrices in the host. Furthermore, three Lys residues in DbpA appear to be critical for the binding of DbpA to decorin. We have now characterized the interaction of DbpA and decorin further by using a synthetic peptide approach. We synthesized a panel of peptides that spanned the DbpA sequence and examined their ability to inhibit the binding of intact DbpA to decorin. From these studies, we identified a decorin-binding peptide that lost this activity if the sequence was either scrambled or if a critical Lys residue was chemically modified. A minimal decorin-binding peptide was identified by examining a set of truncated peptides. One peptide is proposed to contain the primary decorin-binding site in DbpA. By comparing the amino acid sequences of 29 different DbpA homologs from different B. burgdorferi sensu lato isolates, we discovered that the identified decorin-binding sequence was quite variable. Therefore, we synthesized a new panel of peptides containing the putative decorin-binding sequence of the different DbpA homologs. All of these peptides were active in our decorin-binding assay, and consensus decorin binding motifs are discussed.

Publication Types:
PMID: 12761224 [PubMed - indexed for MEDLINE]

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'Lyme disease': ancient engine of an unrecognized borreliosis pandemic?

Harvey WT, Salvato P.

Diversified Medical Practices, Texas, Houston, USA. wth928@aol.com

Unexpectedly we have found large numbers of chronically ill Borrelia burgdorferi PCR- and seropositive patients in Houston, Texas, a zoonotically 'non-endemic' area. In order to understand this finding prior to sufficient data availability, we chose to examine critically currently accepted but troublesome 'Lyme disease' concepts. Our method was to analyze each foundation 'Lyme disease' premise within the context of available medical and veterinary literature, then to reconstruct the disease model consistent with the preponderance of that data. We find the present conceptualization of the illness seriously truncated, with a high likelihood of two distinct but connected forms of human B. burgdorferi infection. The yet-unrecognized form appears to have a broader clinical presentation, wider geographic distribution, and vastly greater prevalence. We conclude that 'Lyme disease' currently acknowledges only its zoonosis arm and is a limited conceptualization of a far more pervasive and unrecognized infection state that must be considered a global epidemic.

PMID: 12710914 [PubMed - indexed for MEDLINE]

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Expression of a luxS gene is not required for Borrelia burgdorferi infection of mice via needle inoculation.

Hübner A, Revel AT, Nolen DM, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

The luxS gene product is an integral component of LuxS/autoinducer-2 (AI-2) quorum-sensing systems in bacteria. A putative luxS gene was expressed at comparable levels by Borrelia burgdorferi strain 297 cultivated either in vitro or in dialysis membrane chambers implanted in rat peritoneal cavities. Although the borrelial luxS gene functionally complemented a LuxS deficiency in Escherichia coli DH5 alpha, AI-2-like activity could not be detected within B. burgdorferi culture supernatants or concentrated cell lysates. Finally, a luxS-deficient mutant of B. burgdorferi was infectious at wild-type levels when it was intradermally needle inoculated into mice, indicating that expression of luxS probably is not required for infectivity but, at the very least, is not essential for mammalian host adaptation. Our findings also challenge the notion that a LuxS/AI-2 quorum-sensing system is operative in B. burgdorferi.

Publication Types:
PMID: 12704164 [PubMed - indexed for MEDLINE]

PMCID: PMC153291


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A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host.

Purser JE, Lawrenz MB, Caimano MJ, Howell JK, Radolf JD, Norris SJ.

Graduate School of Biomedical Sciences and Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston, MSB 2.278, 6431 Fannin St., Houston, TX 77225, USA.

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.

Publication Types:
PMID: 12694619 [PubMed - indexed for MEDLINE]

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Characterization of the vls antigenic variation loci of the Lyme disease spirochaetes Borrelia garinii Ip90 and Borrelia afzelii ACAI.

Wang D, Botkin DJ, Norris SJ.

Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, PO Box 20708, Houston, TX 77225-0708, USA.

The vls locus of Borrelia burgdorferi B31 consists of 15 silent cassettes and one expression site (vlsE), and the presence of the encoding plasmid lp28-1 correlates with high infectivity. Recombination between the expression cassette and silent cassettes occurs in vivo, and this process may enable B. burgdorferi to evade the immune response. To determine the characteristics of the vls loci in other Borrelia strains, we have cloned and characterized the vls silent cassette loci of Borrelia garinii Ip90 and Borrelia afzelii ACAI, consisting of 11 vls silent cassettes and 14 vls silent cassettes respectively. The silent cassettes share 90-97% nucleotide sequence identity with one another within the Ip90 vls locus and 84-91% within the ACAI vls locus. In both organisms, the silent cassettes resemble the B31 Vls sequences in overall amino acid similarity (50-65%) and in the presence of six variable regions interspersed between six relatively invariant regions. The vlsE expression sites of these two strains have not been isolated, but transcripts of vlsE were detected by reverse transcriptase-polymerase chain reaction for both Ip90 and ACAI. In addition, the occurrence of sequence variation within the vlsE cassette region of these transcripts was verified. This study indicates that the vls loci present in B. garinii Ip90 and B. afzelii ACAI have characteristics similar to those found in B. burgdorferi B31.

Publication Types:
PMID: 12603744 [PubMed - indexed for MEDLINE]

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Decreased electroporation efficiency in Borrelia burgdorferi containing linear plasmids lp25 and lp56: impact on transformation of infectious B. burgdorferi.

Lawrenz MB, Kawabata H, Purser JE, Norris SJ.

Graduate School of Biomedical Sciences, Department of Pathology and Laboratory Medicine, Medical School, University of Texas Health Science Center, Houston, Texas 77225-0708, USA.

The presence of the linear plasmids lp25 and lp56 of Borrelia burgdorferi B31 was found to dramatically decrease the rate of transformation by electroporation with the shuttle vector pBSV2, an autonomously replicating plasmid that confers kanamycin resistance (P. E. Stewart, R. Thalken, J. L. Bono, and P. Rosa, Mol. Microbiol. 39:714-721, 2001). B. burgdorferi B31 clones had transformation efficiencies that were either low, intermediate, or high, and this phenotype correlated with the presence or absence of lp25 and lp56. Under the conditions utilized in this study, no transformants were detected in clones that contained both lp25 and lp56; the few kanamycin-resistant colonies isolated did not contain pBSV2, indicating that the resistance was due to mutation. Intermediate electroporation rates (10 to 200 colonies per micro g of DNA) were obtained with B31 clones that were either lp25(-) and lp56(+) or lp25(+) and lp56(-). Clones in this group that initially contained lp25 lacked this plasmid in pBSV2 transformants, a finding consistent with selective transformation of lp25(-) variants. High transformation rates (>1,000 colonies per micro g of DNA) occurred in clones that lacked both lp25 and lp56. Sequence analysis indicated that lp25 and lp56 contain genes that may encode restriction and/or modification systems that could result in the low transformation rates obtained with strains containing these plasmids. The previously reported correlation between lp25 and infectivity in mice, coupled with the barrier lp25 presents to transformation, may explain the difficulty in obtaining virulent transformants of B. burgdorferi.

Publication Types:
PMID: 12183522 [PubMed - indexed for MEDLINE]

PMCID: PMC128261


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Crystal structure of Lyme disease variable surface antigen VlsE of Borrelia burgdorferi.

Eicken C, Sharma V, Klabunde T, Lawrenz MB, Hardham JM, Norris SJ, Sacchettini JC.

Center for Structural Biology, Texas A&M University, College Station, Texas 77843-2128, USA.

VlsE is an outer surface lipoprotein of Borrelia burgdorferi that undergoes antigenic variation through an elaborate gene conversion mechanism and is thought to play a major role in the immune response to the Lyme disease borellia. The crystal structure of recombinant variant protein VlsE1 at 2.3-A resolution reveals that the six variable regions form loop structures that constitute most of the membrane distal surface of VlsE, covering the predominantly alpha-helical, invariant regions of the protein. The surface localization of the variable amino acid segments appears to protect the conserved regions from interaction with antibodies and hence may contribute to immune evasion.

Publication Types:
PMID: 11923306 [PubMed - indexed for MEDLINE]

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DNA microarray analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete.

Revel AT, Talaat AM, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

DNA microarrays were used to survey the adaptive genetic responses of Borrelia burgdorferi (Bb) B31, the Lyme disease spirochete, when grown under conditions analogous to those found in unfed ticks (UTs), fed ticks (FTs), or during mammalian host adaptation (Bb in dialysis membrane chambers implanted in rats). Microarrays contained 95.4% of the predicted B31 genes, 150 (8.6%) of which were differentially regulated (changes of > or = 1.8-fold) among the three growth conditions. A substantial proportion (46%) of the differentially regulated genes encoded proteins with predicted export signals (29% from predicted lipoproteins), emphasizing the importance to Bb of modulating its extracellular proteome. For B31 cultivated at the more restrictive UT condition, microarray data provided evidence of a bacterial stringent response and factors that restrict cell division. A large proportion of genes were responsive to the FT growth condition, wherein increased temperature and reduced pH were prominent environmental parameters. A surprising theme, supported by cluster analysis, was that many of the gene expression changes induced during the FT growth condition were transient and largely tempered as B31 adapted to the mammalian host, suggesting that once Bb gains entry and adapts to mammalian tissues, fewer differentially regulated genes are exploited. It therefore would seem that although widely dissimilar, the UT and dialysis membrane chamber growth conditions promote more static patterns of gene expression in Bb. The microarray data thus provide a basis for formulating new testable hypotheses regarding the life cycle of Bb and attaining a more complete understanding of many aspects of Bb's complex parasitic strategies.

Publication Types:
PMID: 11830671 [PubMed - indexed for MEDLINE]

PMCID: PMC122230


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Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway.

Hübner A, Yang X, Nolen DM, Popova TG, Cabello FC, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

RpoS and RpoN are two alternative sigma factors typically associated with general stress responses in bacteria. To date, there has been no experimental evidence that RpoS and RpoN can directly control the expression of one another. Herein, using a combined strategy of gene disruption and genetic complementation targeting rpoN and rpoS in Borrelia burgdorferi strain 297, we describe a regulatory network for B. burgdorferi. In this network, RpoN controls the expression of RpoS, which, in turn, governs the expression of two important membrane lipoproteins, outer surface protein C and decorin-binding protein A, and likely other proteins of B. burgdorferi. Our findings provide a foundation for elucidating further key regulatory networks that potentially impact many aspects of B. burgdorferi's parasitic strategy, host range, and virulence expression.

Publication Types:
PMID: 11675503 [PubMed - indexed for MEDLINE]

PMCID: PMC60121


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Genetic heterogeneity of Borrelia burgdorferi sensu lato in the southern United States based on restriction fragment length polymorphism and sequence analysis.

Lin T, Oliver JH Jr, Gao L, Kollars TM Jr, Clark KL.

Institute of Arthropodology and Parasitology, Department of Biology, Georgia Southern University, Statesboro, Georgia 30460-8056, USA.

Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of rrf-rrl intergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in the B. burgdorferi sensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related to Borrelia garinii. The MOK-3a group separated into a distinct deep branch in the B. andersonii lineage. PCR-RFLP analysis results and the results of sequence analyses of the rrf-rrl intergenic spacer confirm that greater genetic heterogeneity exists among B. burgdorferi sensu lato strains isolated from the southern United States than among strains isolated from the northern United States. The B. andersonii genospecies and its MOK-3a subgroup are associated with the I. dentatus-cottontail rabbit enzootic cycle, but I. scapularis was also found to harbor a strain of this genospecies. Strains that appear to be B. bissettii in our study were isolated from I. minor and the cotton mouse, cotton rat, and wood rat. The B. burgdorferi sensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.

Publication Types:
PMID: 11427560 [PubMed - indexed for MEDLINE]

PMCID: PMC88176


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The effect of UV irradiation on infection of mice with Borrelia burgdorferi.

Brown EL, Ullrich SE, Pride M, Kripke ML.

Department of Immunology, P.O. 178, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

These studies addressed the hypothesis that UV radiation (UVR) could affect immune responses in mice infected with Borrelia burgdorferi. Immunity against the Lyme spirochete B. burgdorferi was studied in a murine model of UV-induced immune suppression. Borrelia-specific cellular and humoral responses were examined following immunosuppressive doses of UVR. Low-passage Borrelia were injected intradermally at the base of the tail following irradiation. At various time points after infection the blood was cultured for the presence of Borrelia and the serum analyzed for Borrelia-specific antibodies. Two weeks after infection one hind-limb joint was cultured for the presence of spirochetes and the contralateral joint was examined histologically for arthritis formation. The results demonstrated that UV irradiation, administered at the site of infection or at a distant site, suppressed Borrelia-specific cellular and humoral responses in infected mice. Suppression of delayed-type hypersensitivity and antibody responses to UV was abrogated by administration of anti-interleukin (IL)-10 after UV irradiation. In addition, UV irradiation altered the dissemination pattern of the bacteria from the skin into the blood and exacerbated arthritis when compared with unirradiated controls. From these studies we concluded that UV irradiation can modulate the immune response to Borrelia and exacerbate the subsequent arthritic component of Lyme disease in mice. Furthermore, our studies suggest that IL-10 is in part responsible for the suppression of both cellular and humoral responses in addition to playing a role in the development of Lyme arthritis.

Publication Types:
PMID: 11367577 [PubMed - indexed for MEDLINE]

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Influence of cultivation media on genetic regulatory patterns in Borrelia burgdorferi.

Yang X, Popova TG, Goldberg MS, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75390, USA.

Barbour-Stoenner-Kelly II (BSKII) medium and BSKH medium both are routinely used for the cultivation of Borrelia burgdorferi. However, heretofore there have been no studies to compare how these two media affect gene expression patterns in virulent B. burgdorferi. In the present study, we found that some B. burgdorferi strain 297 genes (e.g., ospA, mlp-7A, mlp-8, p22, and lp6.6) that typically are regulated by temperature or pH displayed their predicted pattern of expression when B. burgdorferi was cultivated in BSKH medium; this was not true when spirochetes were cultivated in conventional BSKII medium. The results suggest that BSKH medium is superior to BSKII medium for gene expression studies with B. burgdorferi.

Publication Types:
PMID: 11349092 [PubMed - indexed for MEDLINE]

PMCID: PMC98485


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Resistance to Lyme disease in decorin-deficient mice.

Brown EL, Wooten RM, Johnson BJ, Iozzo RV, Smith A, Dolan MC, Guo BP, Weis JJ, Höök M.

The Center for Extracellular Matrix Biology, Texas A&M University System Health Science Center, Albert B. Alkek Institute of Biosciences and Technology, Houston, Texas 77030, USA. ebrown@ibt.tamu.edu

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Publication Types:
PMID: 11285303 [PubMed - indexed for MEDLINE]

PMCID: PMC199574


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VraA (BBI16) protein of Borrelia burgdorferi is a surface-exposed antigen with a repetitive motif that confers partial protection against experimental Lyme borreliosis.

Labandeira-Rey M, Baker EA, Skare JT.

Department of Medical Microbiology and Immunology, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.

We have previously described the expression cloning of nine Borrelia burgdorferi antigens, using rabbit serum enriched for antibodies specific for infection-associated antigens, and determined that seven of these antigens were associated with infectious B. burgdorferi strain B31. One of these infection-associated antigens encoded a 451-amino-acid putative lipoprotein containing 21 consecutive and invariant 9-amino-acid repeat sequences near the amino terminus that we have designated VraA for virulent strain-associated repetitive antigen A. The vraA locus (designated BBI16 by The Institute for Genomic Research) maps to one of the 28-kb linear plasmids (designated lp28-4) that is not present in noninfectious strain B31 isolates. Subsequent PCR analysis of clonal isolates of B. burgdorferi B31 from infected mouse skin revealed a clone that lacked only lp28-4. Southern blot and Western blot analyses indicated that the lp28-4 and VraA proteins, respectively, were missing from this clone. We have also determined that VraA is a surface-exposed protein based on protease accessibility assays of intact whole cells. Furthermore, vraA expression is modestly derepressed when cells are grown at 37 degrees C relative to cells grown at 32 degrees C, suggesting that VraA is, in part, a temperature-inducible antigen. Homologues cross-reactive to B. burgdorferi B31 VraA, most with different molecular masses, were identified in several B. burgdorferi sensu lato isolates, including B. andersonii, suggesting that the immunogenic epitope(s) present in strain B31 VraA is conserved between Borrelia spp. In protection studies, only 8.3% of mice (1 of 12) immunized with full-length recombinant VraA fused to glutathione S-transferase (GST) were susceptible to infectious challenge with 10(2) B. burgdorferi strain B31, whereas naive mice or mice immunized with GST alone were infected 40% or 63 to 67% (depending on tissues assayed) of the time, respectively. As such, the partial protection elicited by VraA immunization provides an additional testable vaccine candidate to help protect against Lyme borreliosis.

Publication Types:
PMID: 11179306 [PubMed - indexed for MEDLINE]

PMCID: PMC98035


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Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi.

Eicken C, Sharma V, Klabunde T, Owens RT, Pikas DS, Höök M, Sacchettini JC.

Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.

The outer surface protein C (OspC) is one of the major host-induced antigens of Borrelia burgdorferi, the causative agent of Lyme disease. We have solved the crystal structure of recombinant OspC to a resolution of 2.5 A. OspC, a largely alpha-helical protein, is a dimer with a characteristic central four-helical bundle formed by association of the two longest helices from each subunit. OspC is very different from OspA and similar to the extracellular domain of the bacterial aspartate receptor and the variant surface glycoprotein from Trypanosoma brucei. Most of the surface-exposed residues of OspC are highly variable among different OspC isolates. The membrane proximal halves of the two long alpha-helices are the only conserved regions that are solvent accessible. As vaccination with recombinant OspC has been shown to elicit a protective immune response in mice, these regions are candidates for peptide-based vaccines.

Publication Types:
PMID: 11139584 [PubMed - indexed for MEDLINE]

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Decreased infectivity in Borrelia burgdorferi strain B31 is associated with loss of linear plasmid 25 or 28-1.

Labandeira-Rey M, Skare JT.

Department of Medical Microbiology and Immunology, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.

Previous reports indicated a correlation between loss of plasmids and decreased infectivity of Borrelia burgdorferi strain B31, suggesting that plasmids may encode proteins that are required for pathogenesis. In this study, we expand on this correlation. Using the B. burgdorferi genomic sequence, we designed primers specific for each plasmid, and by using PCR we catalogued 11 linear and 2 circular plasmids from 49 clonal isolates of a mid-passage B. burgdorferi strain B31, initially derived from infected mouse skin, and 20 clones obtained from mouse skin infected with a low-passage isolate of B. burgdorferi strain B31. Among the 69 clones analyzed, nine distinct genotypes were identified relative to wild-type B. burgdorferi strain B31. Among the nine clonal genotypes obtained, only the 9-kb circular plasmid (cp9), the 25-kb linear plasmid (lp25), and either the 28-kb linear plasmid 1 or 4 (lp28-1 and lp28-4, respectively) were missing, in different combinations. We compared the infectivity of the wild-type strain, containing all known B. burgdorferi plasmids, with those of single mutants lacking either lp28-1, lp28-4, or lp25 and a double mutant missing both cp9 and lp28-1. The infectivity data indicated that B. burgdorferi strain B31 cells lacking lp28-4 were modestly attenuated in all tissues analyzed, whereas samples missing lp25 were completely attenuated in all tissues, even at the highest inoculum tested. Isolates without lp28-1 infected the joint tissue yet were not able to infect other tissues as effectively. In addition, we have observed a selection in vivo in the skin, bladder, and joint for cells containing lp25 and in the skin and bladder for cells containing lp28-1, indicating that lp25 and lp28-1 encode proteins required for colonization and short-term maintenance in these mammalian tissues. In contrast, there was no selection in the joint for cells containing lp28-1, suggesting that genes on lp28-1 are not required for colonization of B. burgdorferi within the joint. These observations imply that the dynamic nature of the B. burgdorferi genome may provide the genetic heterogeneity necessary for survival in the diverse milieus that this pathogen occupies in nature and may contribute to tropism in certain mammalian host tissues.

Publication Types:
PMID: 11119536 [PubMed - indexed for MEDLINE]

PMCID: PMC97902


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Correlation between plasmid content and infectivity in Borrelia burgdorferi.

Purser JE, Norris SJ.

Departments of Pathology and Laboratory Medicine, and Microbiology and Molecular Genetics, Medical School, and Graduate School of Biomedical Sciences, University of Texas-Houston Health Science Center, P.O. Box 20708, Houston, TX 77225, USA.

Infectivity-associated plasmids were identified in Borrelia burgdorferi B31 by using PCR to detect each of the plasmids in a panel of 19 clonal isolates. The clones exhibited high-, low-, and intermediate-infectivity phenotypes based on their frequency of isolation from needle-inoculated C3H/HeN mice. Presence or absence of 21 of the 22 plasmids was determined in each of the clones by using PCR primers specific for regions unique to each plasmid, as identified in the recently available genome sequence. Southern blot hybridization results were used to confirm the PCR results in some cases. Plasmid lp25 exhibited a direct correlation with infectivity in that it was consistently present in all clones of high or intermediate infectivity and was absent in all low-infectivity clones. lp28-1, containing the vmp-like sequence locus, also correlated with infectivity; all clones that lacked lp28-1 but contained lp25 had an intermediate infectivity phenotype, in which infection was primarily restricted to the joints. Plasmids cp9, cp32-3, lp21, lp28-2, lp28-4, and lp56 apparently are not required for infection in this model, because clones lacking these plasmids exhibited a high-infectivity phenotype. Plasmids cp26, cp32-1, cp32-2 and/or cp32-7, cp32-4, cp32-6, cp32-8, cp32-9, lp17, lp28-3, lp36, lp38, and lp54 were consistently present in all clones examined. On the basis of these results, lp25 and lp28-1 appear to encode virulence factors important in the pathogenesis of B. burgdorferi B31.

Publication Types:
PMID: 11106398 [PubMed - indexed for MEDLINE]

PMCID: PMC17667


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The many faces of Borrelia burgdorferi.

Seshu J, Skare JT.

Department of Medical Microbiology and Immunology, The Texas A&M University System Health Science Center, College Station 77843-1114, USA.

In this review we describe several genetic regulatory mechanisms adopted by the agent of Lyme disease, Borrelia burgdorferi, to sense and adapt to different host and environmental conditions either in vitro or in vivo. This regulation results in the increased or decreased synthesis of several proteins whose levels are believed to play key roles in the ability of B. burgdorferi to cycle between both arthropod and mammalian hosts. Moreover, the differential synthesis of these proteins serves to modulate the response of B. burgdorferito signals in the requisite host and may also, in some cases, function as virulence determinants of this spirochete. Elucidation of these mechanisms will help in the understanding of the pathogenicity of B. burgdorferi as well as aid in identifying proteins that are important during different stages of infection.

Publication Types:
PMID: 11075919 [PubMed - indexed for MEDLINE]

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Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi.

Yang X, Goldberg MS, Popova TG, Schoeler GB, Wikel SK, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas South-western Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75235, USA.

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC-like (e.g. ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s). The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.

Publication Types:
PMID: 10998177 [PubMed - indexed for MEDLINE]

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Decorin-binding protein A (DbpA) of Borrelia burgdorferi is not protective when immunized mice are challenged via tick infestation and correlates with the lack of DbpA expression by B. burgdorferi in ticks.

Hagman KE, Yang X, Wikel SK, Schoeler GB, Caimano MJ, Radolf JD, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

Previous studies showed that decorin-binding protein A (DbpA) of Borrelia burgdorferi was a protective immunogen in the murine model of Lyme borreliosis when mice were challenged (needle inoculated) intradermally with in vitro-cultivated spirochetes. In the present study, DbpA-immunized C3H/HeJ mice were not protected from infection when infested with Ixodes scapularis nymphs harboring virulent B. burgdorferi 297. This lack of protection correlated with the failure to detect DbpA on B. burgdorferi in ticks, suggesting that DbpA is not available as a target for bactericidal antibodies in serum when B. burgdorferi-infected ticks take their blood meal from an immunized host. The failure of DbpA immunization to protect tick-challenged mice contradicts the results of earlier needle inoculation vaccination experiments and suggests that DbpA may not be suitable as a Lyme disease vaccine.

Publication Types:
PMID: 10899883 [PubMed - indexed for MEDLINE]

PMCID: PMC98430


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Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi.

Lawrenz MB, Hardham JM, Owens RT, Nowakowski J, Steere AC, Wormser GP, Norris SJ.

Departments of Pathology and Laboratory Medicine and Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas 77030, USA.

VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.

Publication Types:
PMID: 10565921 [PubMed - indexed for MEDLINE]

PMCID: PMC85865


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Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family.

Yang X, Popova TG, Hagman KE, Wikel SK, Schoeler GB, Caimano MJ, Radolf JD, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

We previously reported on the existence of a family of lipoprotein genes, designated 2.9 lipoprotein genes, encoded in at least seven versions on the circular (supercoiled) cp32 and cp18 plasmids of Borrelia burgdorferi 297. A distinguishing feature of the 2.9 lipoproteins were highly similar signal sequences but variable mature polypeptides that segregated into two antigenic classes. Further screenings of B. burgdorferi 297 genomic libraries led to the identification of three additional 2.9 lipoprotein genes, renamed herein mlp, for multicopy lipoprotein genes. Computer analyses and immunoblotting revealed that Mlp-9 segregated with the antigenic class I lipoproteins, whereas Mlp-8 and Mlp-10 were members of class II. Northern blotting showed that all three of the mlp genes were expressed when B. burgdorferi was cultivated in vitro at 34 degrees C, although mlp-9 and mlp-10 transcripts were expressed at very low levels. Additional combined immunoblotting and comparative reverse transcription-PCR analyses performed on borreliae cultivated in vitro at 23, 34, or 37 degrees C indicated that although Mlp-8 was substantially more abundant than Mlp-9 or Mlp-10, all three of the mlp genes were upregulated during B. burgdorferi replication at 37 degrees C. Expression of the same three lipoproteins was further enhanced upon growth of the spirochetes within dialysis membrane chambers (DMCs) implanted intraperitoneally in rats (i.e., spirochetes in a mammalian host-adapted state), suggesting that temperature alone did not account for maximal upregulation of the mlp genes. That certain mlp genes are likely expressed during the growth of B. burgdorferi in mammalian tissues was supported by findings of antibodies against all three Mlp lipoproteins in mice after challenge with Ixodes scapularis nymphs harboring B. burgdorferi 297. The combined data suggest that as opposed to being differentially expressed in any reciprocal fashion (e.g., OspA/OspC), at least three mlp genes are simultaneously upregulated by temperature (37 degrees C) and some other mammalian host factor(s). The findings have importance not only for understanding alternative modes of differential antigen expression by B. burgdorferi but also for assessing whether one or more of the Mlp lipoproteins represent new candidate vaccinogens for Lyme disease.

Publication Types:
PMID: 10531261 [PubMed - indexed for MEDLINE]

PMCID: PMC96987


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Adherence of Borrelia burgdorferi. Identification of critical lysine residues in DbpA required for decorin binding.

Brown EL, Guo BP, O'Neal P, Höök M.

Center for Extracellular Matrix Biology, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA.

Borrelia burgdorferi, the causative agent of Lyme disease, expresses on its surface two decorin binding adhesins, DbpA and DbpB. Previous studies have demonstrated that vaccination of mice with DbpA provided protection against challenge with heterologous Borrelia strains despite considerable sequence variability among DbpA in these strains. We have now examined the importance of individual amino acid residues in DbpA for decorin binding. We demonstrated that chemical modification of lysine residues resulted in loss of ligand binding activity. Of the 27 lysine residues in native DbpA from strain 297, 6 are present in most and 5 are conserved in all 30 DbpA sequences examined so far. Analysis of recombinant DbpA in which individual lysine residues have been mutated to alanine suggested that three of the conserved residues distributed throughout the DbpA sequence are required for decorin binding. These mutants lost their ability to bind decorin in Western ligand blot assay and bound reduced amounts of decorin in an ELISA. Furthermore, these mutant DbpA proteins did not inhibit the adherence of B. burgdorferi to a decorin substrata, and they did not recognize decorin in an extracellular matrix established by human fibroblast cultures. We conclude that the three lysine residues Lys-82, Lys-163, and Lys-170 are crucial for the binding of DbpA to decorin.

Publication Types:
PMID: 10473582 [PubMed - indexed for MEDLINE]

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Cloning and molecular characterization of plasmid-encoded antigens of Borrelia burgdorferi.

Skare JT, Foley DM, Hernandez SR, Moore DC, Blanco DR, Miller JN, Lovett MA.

Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station, Texas 77843-1114, USA. jskare@tamu.edu

Thirteen independent clones that encode Borrelia burgdorferi antigens utilizing antiserum from infection-immune rabbits were identified. The serum was adsorbed against noninfectious B. burgdorferi B31 to enrich for antibodies directed against either infection-associated antigens of B. burgdorferi B31 or proteins preferentially expressed during mammalian infection. The adsorption efficiency of the immune rabbit serum (IRS) was assessed by Western immunoblot analysis with protein lysates derived from infectious and noninfectious B. burgdorferi B31. The adsorbed IRS was used to screen a B. burgdorferi expression library to identify immunoreactive phage clones. Clones were then expressed in Escherichia coli and subsequently analyzed by Western blotting to determine the molecular mass of the recombinant B. burgdorferi antigens. Southern blot analysis of the 13 clones indicated that 10 contained sequences unique to infectious B. burgdorferi. Nucleotide sequence analysis indicated that the 13 clones were composed of 9 distinct genetic loci and that all of the genes identified were plasmid encoded. Five of the clones carried B. burgdorferi genes previously identified, including those encoding decorin binding proteins A and B (dbpAB), a rev homologue present on the 9-kb circular plasmid (cp9), a rev homologue from the 32-kb circular plasmid (cp32-6), erpM, and erpX. Additionally, four previously uncharacterized loci with no known homologues were identified. One of these unique clones encoded a 451-amino-acid lipoprotein with 21 consecutive, invariant 9-amino-acid repeats near the amino terminus that we have designated VraA (for "virulent strain-associated repetitive antigen A"). Since all the antigens identified are recognized by serum from infection immune rabbits, these antigens represent potential vaccine candidates and, based on the identification of dbpAB in this screen, may also be involved in pathogenic processes operative in Lyme borreliosis.

Publication Types:
PMID: 10456881 [PubMed - indexed for MEDLINE]

PMCID: PMC96759


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Activation of human monocytic cells by Borrelia burgdorferi and Treponema pallidum is facilitated by CD14 and correlates with surface exposure of spirochetal lipoproteins.

Sellati TJ, Bouis DA, Caimano MJ, Feulner JA, Ayers C, Lien E, Radolf JD.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235, USA.

Here we examined the involvement of CD14 in monocyte activation by motile Borrelia burgdorferi and Treponema pallidum. B. burgdorferi induced secretion of IL-8 by vitamin D3-matured THP-1 cells, which was inhibited by a CD14-specific mAb known to block cellular activation by LPS and the prototypic spirochetal lipoprotein, outer surface protein A. Enhanced responsiveness to B. burgdorferi also was observed when THP-1 cells were transfected with CD14. Because borreliae within the mammalian host and in vitro-cultivated organisms express different lipoproteins, experiments also were performed with "host-adapted" spirochetes grown within dialysis membrane chambers implanted into the peritoneal cavities of rabbits. Stimulation of THP-1 cells by host-adapted organisms was CD14 dependent and, interestingly, was actually greater than that observed with in vitro-cultivated organisms grown at either 34 degrees C or following temperature shift from 23 degrees C to 37 degrees C. Consistent with previous findings that transfection of Chinese hamster ovary cells with CD14 confers responsiveness to LPS but not to outer surface protein A, B. burgdorferi failed to stimulate CD14-transfected Chinese hamster ovary cells. T. pallidum also activated THP-1 cells in a CD14-dependent manner, although its stimulatory capacity was markedly less than that of B. burgdorferi. Moreover, cell activation by motile T. pallidum was considerably less than that induced by treponemal sonicates. Taken together, these findings support the notion that lipoproteins are the principle component of intact spirochetes responsible for monocyte activation, and they indicate that surface exposure of lipoproteins is an important determinant of a spirochetal pathogen's proinflammatory capacity.

Publication Types:
PMID: 10438943 [PubMed - indexed for MEDLINE]

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Isolation of a new subspecies, Bartonella vinsonii subsp. arupensis, from a cattle rancher: identity with isolates found in conjunction with Borrelia burgdorferi and Babesia microti among naturally infected mice.

Welch DF, Carroll KC, Hofmeister EK, Persing DH, Robison DA, Steigerwalt AG, Brenner DJ.

Laboratory Corporation of America, Dallas, Texas 75230, USA. da_welchd@rodeo.biomed.com

Bacteremia with fever due to a novel subspecies of Bartonella vinsonii was found in a cattle rancher. The subspecies shared major characteristics of the genus Bartonella in terms of most biochemical features and cellular fatty acid profile, but it was distinguishable from other subspecies of B. vinsonii by good growth on heart infusion agar supplemented with X factor and by its pattern of enzymatic hydrolysis of peptide substrates. DNA relatedness studies verified that the isolate belonged to the genus Bartonella and that it was genotypically related to B. vinsonii. The highest level of relatedness was observed with recently characterized strains from naturally infected mice that were coinfected with Borrelia burgdorferi and Babesia microti. We propose the name Bartonella vinsonii subsp. arupensis subsp. nov. as the new subspecies to accommodate these human and murine isolates.

Publication Types:
PMID: 10405408 [PubMed - indexed for MEDLINE]

PMCID: PMC85292


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Decorin-binding adhesins from Borrelia burgdorferi.

Guo BP, Brown EL, Dorward DW, Rosenberg LC, Höök M.

Albert B. Alkek Institute of Biosciences and Technology and the Department of Biochemistry and Biophysics, Texas A & M University, Houston 77030, USA.

Lyme disease is a tick-transmitted infection caused by the spirochete Borrelia burgdorferi. Ticks deposit B. burgdorferi into the dermis of the host, where they eventually become associated with collagen fibres. We demonstrated previously that B. burgdorferi is unable to bind collagen, but can bind the collagen-associated proteoglycan decorin and expresses decorin-binding proteins (Dbps). We have now cloned and sequenced two genes encoding the proteins, DbpA and DbpB, which have a similar structure, as revealed by circular dichroism (CD) spectroscopy of recombinant proteins. Competition experiments revealed a difference in binding specificity between DbpA and DbpB. Western blot analysis of proteinase K-treated intact B. burgdorferi and transmission electron microscopy studies using antibodies raised against recombinant Dbps demonstrated that these proteins are surface exposed. DbpA effectively inhibits the attachment of B. burgdorferi to a decorin substrate, whereas DbpB had a marginal effect, suggesting a difference in substrate specificity between the two Dbps. Polystyrene beads coated with DbpA adhered to a decorin-containing extracellular matrix produced by cultured skin fibroblasts, whereas beads coated with OspC did not. Taken together, these data suggest that Dbps are adhesins of the MSCRAMM (microbial surface component-recognizing adhesive matrix molecule) family, which mediate B. burgdorferi attachment to the extracellular matrix of the host.

Publication Types:
PMID: 10094620 [PubMed - indexed for MEDLINE]

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Molecular and evolutionary analysis of Borrelia burgdorferi 297 circular plasmid-encoded lipoproteins with OspE- and OspF-like leader peptides.

Akins DR, Caimano MJ, Yang X, Cerna F, Norgard MV, Radolf JD.

Departments of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

We previously described two OspE and three OspF homologs in Borrelia burgdorferi 297 (D. R. Akins, S. F. Porcella, T. G. Popova, D. Shevchenko, S. I. Baker, M. Li, M. V. Norgard, and J. D. Radolf, Mol. Microbiol. 18:507-520, 1995; D. R. Akins, K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf, J. Clin. Investig. 101:2240-2250, 1998). In this study, we characterized four additional lipoproteins with OspE/F-like leader peptides (Elps) and demonstrated that all are encoded on plasmids homologous to cp32 and cp18 from the B31 and N40 strains, respectively. Statistical analysis of sequence similarities using the binary comparison algorithm revealed that the nine lipoproteins from strain 297, as well as the OspE, OspF, and Erp proteins from the N40 and B31 strains, fall into three distinct families. Based upon the observation that these lipoproteins all contain highly conserved leader peptides, we now propose that the ancestors of each of the three families arose from gene fusion events which joined a common N terminus to unrelated proteins. Additionally, further sequence analysis of the strain 297 circular plasmids revealed that rearrangements appear to have played an important role in generating sequence diversity among the members of these three families and that recombinational events in the downstream flanking regions appear to have occurred independently of those within the lipoprotein-encoding genes. The association of hypervariable regions with genes which are differentially expressed and/or subject to immunological pressures suggests that the Lyme disease spirochete has exploited recombinatorial processes to foster its parasitic strategy and enhance its immunoevasiveness.

Publication Types:
PMID: 10024606 [PubMed - indexed for MEDLINE]

PMCID: PMC96492


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Lyme disease associated with unilateral interstitial keratitis.

Miyashiro MJ, Yee RW, Patel G, Ruiz RS.

Department of Ophthalmology and Visual Science, The University of Texas Medical School at Houston, 77030, USA.

PURPOSE: To report a case of Lyme disease that presented with a single nummular unilateral interstitial keratitis. METHODS: Case report and review of the literature. RESULTS: A 57-year-old black man who had contact with freshly killed deer had a chief complaint of foreign-body sensation in his right eye (OD) that had been diagnosed and treated for herpes simplex stromal keratitis. The patient underwent a systemic workup for interstitial keratitis. All results including RPR and MHA-TP were negative except for Lyme antibody titer (enzyme-linked immunosorbent assay [ELISA]) 178 U/ml (normal, <159 U/ml). CONCLUSION: Interstitial keratitis from Lyme disease has been regarded as a bilateral disease in the literature. We present this infrequent ocular manifestation of Lyme disease as a rare single nummular unilateral presentation.

Publication Types:
PMID: 9894947 [PubMed - indexed for MEDLINE]

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A proposal for the reliable culture of Borrelia burgdorferi from patients with chronic Lyme disease, even from those previously aggressively treated.

Phillips SE, Mattman LH, Hulínská D, Moayad H.

Greenwich Hospital, CT 06830, USA.

Since culture of Borrelia burgdorferi from patients with chronic Lyme disease has been an extraordinarily rare event, clarification of the nature of the illness and proving its etiology as infectious have been difficult. A method for reliably and reproducibly culturing B. burgdorferi from the blood of patients with chronic Lyme disease was therefore sought by making a controlled blood culture trial studying 47 patients with chronic Lyme disease. All had relapsed after long-term oral and intravenous antibiotics. 23 patients with other chronic illness formed the control group. Positive cultures were confirmed by fluorescent antibody immuno-electron microscopy using monoclonal antibody directed against Osp A, and Osp A PCR. 43/47 patients (91%) cultured positive. 23/23 controls (100%) cultured negative. Although persistent infection has been, to date, strongly suggested in chronic Lyme disease by positive PCR and antigen capture, there are major problems with these tests. This new method for culturing B. burgdorferi from patients with chronic Lyme disease certainly defines the nature of the illness and establishes that it is of chronic infectious etiology. This discovery should help to reestablish the gold standard in laboratory diagnosis of Lyme disease.

Publication Types:
PMID: 9861561 [PubMed - indexed for MEDLINE]

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Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion.

Zhang JR, Norris SJ.

Department of Pathology and Laboratory Medicine and Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas 77030, USA.

The Lyme disease spirochete Borrelia burgdorferi possesses 15 silent vls cassettes and a vls expression site (vlsE) encoding a surface-exposed lipoprotein. Segments of the silent vls cassettes have been shown to recombine with the vlsE cassette region in the mammalian host, resulting in combinatorial antigenic variation. Despite promiscuous recombination within the vlsE cassette region, the 5' and 3' coding sequences of vlsE that flank the cassette region are not subject to sequence variation during these recombination events. The segments of the silent vls cassettes recombine in the vlsE cassette region through a unidirectional process such that the sequence and organization of the silent vls loci are not affected. As a result of recombination, the previously expressed segments are replaced by incoming segments and apparently degraded. These results provide evidence for a gene conversion mechanism in VlsE antigenic variation.

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PMID: 9673251 [PubMed - indexed for MEDLINE]

PMCID: PMC108404


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Erratum in:
  • Infect Immun 1999 Jan;67(1):468.

Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi.

Zhang JR, Norris SJ.

Department of Pathology and Laboratory Medicine and Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas 77030, USA.

The Lyme disease agent, Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host. vlsE, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in B. burgdorferi B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice. In this study, we examined the kinetics of vlsE sequence variation in C3H/HeN mice at 4, 7, 14, 21, and 28 days and at 7 and 12 months postinfection. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection. The sequence changes were consistent with sequential recombination of segments from multiple silent vls cassette sites into the vlsE expression site. No vlsE sequence changes were detected in organisms cultured in vitro for up to 84 days. These results indicate that vlsE recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation. The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.

Publication Types:
PMID: 9673250 [PubMed - indexed for MEDLINE]

PMCID: PMC108403


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Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a CD14-dependent pathway distinct from that used by lipopolysaccharide.

Sellati TJ, Bouis DA, Kitchens RL, Darveau RP, Pugin J, Ulevitch RJ, Gangloff SC, Goyert SM, Norgard MV, Radolf JD.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235, USA.

Lipoproteins of Treponema pallidum and Borrelia burgdorferi possess potent proinflammatory properties and, thus, have been implicated as major proinflammatory agonists in syphilis and Lyme disease. Here we used purified B. burgdorferi outer surface protein A (OspA) and synthetic lipopeptides corresponding to the N-termini of OspA and the 47-kDa major lipoprotein immunogen of T. pallidum to clarify the contribution of CD14 to monocytic cell activation by spirochetal lipoproteins and lipopeptides. As with LPS, mouse anti-human CD14 Abs blocked the activation of 1,25-dihydroxyvitamin D3-matured human myelomonocytic THP-1 cells by OspA and the two lipopeptides. The existence of a CD14-dependent pathway was corroborated by using undifferentiated THP-1 cells transfected with CD14 and peritoneal macrophages from CD14-deficient BALB/c mice. Unlike LPS, cell activation by lipoproteins and lipopeptides was serum independent and was not augmented by exogenous LPS-binding protein. Two observations constituted evidence that LPS and lipoprotein/lipopeptide signaling proceed via distinct transducing elements downstream of CD14: 1) CHO cells transfected with CD14 were exquisitely sensitive to LPS but were lipoprotein/lipopeptide nonresponsive; and 2) substoichiometric amounts of deacylated LPS that block LPS signaling at a site distal to CD14 failed to antagonize activation by lipoproteins and lipopeptides. The combined results demonstrate that spirochetal lipoproteins and lipopeptides use a CD14-dependent pathway that differs in at least two fundamental respects from the well-characterized LPS recognition pathway.

Publication Types:
PMID: 9605148 [PubMed - indexed for MEDLINE]

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Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis.

Hagman KE, Lahdenne P, Popova TG, Porcella SF, Akins DR, Radolf JD, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

Isolated outer membranes of Borrelia burgdorferi were used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [3H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) of B. burgdorferi N40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467-3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of the dbp gene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 and dbp comprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts of B. burgdorferi isolated outer membranes. Mice infected with B. burgdorferi produced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbp operon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.

Publication Types:
PMID: 9596733 [PubMed - indexed for MEDLINE]

PMCID: PMC108255


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A new animal model for studying Lyme disease spirochetes in a mammalian host-adapted state.

Akins DR, Bourell KW, Caimano MJ, Norgard MV, Radolf JD.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

There is now substantial evidence that Borrelia burgdorferi, the Lyme disease spirochete, undergoes major alterations in antigenic composition as it cycles between its arthropod and mammalian hosts. In this report, we cultivated B. burgdorferi 297 within dialysis membrane chambers implanted into the peritoneal cavities of rats to induce antigenic changes similar to those which occur during mammalian infection. Chamber-grown spirochetes, which remained fully virulent, did not express either outer surface protein A or Lp6.6, lipoproteins known to be downregulated after mammalian infection. However, they did, express p21, a well characterized outer surface protein E homologue, which is selectively expressed during infection. SDS-PAGE, two-dimensional gel electrophoresis, and immunoblot analysis revealed that chamber-grown borreliae also expressed uncharacterized proteins not expressed by in vitro-cultivated spirochetes; reactivity with sera from mice chronically infected with B. burgdorferi 297 confirmed that many of these novel proteins are selectively expressed during experimental murine infection. Finally, we used differential display RT-PCR to identify transcripts of other differentially expressed B. burgdorferi genes. One gene (2.9-7lpB) identified with this technique belongs to a family of genes located on homologous 32- and 18-kb circular plasmids. The lipoprotein encoded by 2.9-7lpB was shown to be selectively expressed by chamber-grown spirochetes and by spirochetes during experimental infection. Cultivation of B. burgdorferi in rat peritoneal implants represents a novel system for studying Lyme disease spirochetes in a mammalian host-adapted state.

Publication Types:
PMID: 9593780 [PubMed - indexed for MEDLINE]

PMCID: PMC508812


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Specific Th1 cell lines that confer protective immunity against experimental Borrelia burgdorferi infection in mice.

Pride MW, Brown EL, Stephens LC, Killion JJ, Norris SJ, Kripke ML.

Department of Immunology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.

Although humoral responses to Borrelia burgdorferi (Bb) have been shown to be protective in some animal models of Lyme disease, the role of T cells in this disease is less well understood. This work describes three Bb-specific T cell lines that prevent disease progression in syngeneic mice. The T cell lines were generated in C3H mice immunized with Bb in complete Freund's adjuvant. All lines were Bb-specific, CD4+, TCRalphabeta+, and they proliferated and produced interferon-gamma and interleukin-2 on stimulation with Bb. Injection of the cell lines into naive C3H recipients significantly reduced the number of organisms recoverable from the blood and tissues of infected mice and protected them from developing Bb-induced periarthritis. These studies demonstrated that Th1 cells can confer resistance to Bb infection in susceptible mice and suggested that the timing of this T cell response may be critical for determining disease outcome.

Publication Types:
PMID: 9581797 [PubMed - indexed for MEDLINE]

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The Oms66 (p66) protein is a Borrelia burgdorferi porin.

Skare JT, Mirzabekov TA, Shang ES, Blanco DR, Erdjument-Bromage H, Bunikis J, Bergström S, Tempst P, Kagan BL, Miller JN, Lovett MA.

Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station 77843, USA. jskare@tamu.edu

In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.

Publication Types:
PMID: 9284133 [PubMed - indexed for MEDLINE]

PMCID: PMC175520


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Immunologic and genetic analyses of VmpA of a neurotropic strain of Borrelia turicatae.

Cadavid D, Pennington PM, Kerentseva TA, Bergström S, Barbour AG.

Department of Microbiology, University of Texas Health Science Center at San Antonio, 78284, USA.

In mice infected with serotype A but not serotype B of the relapsing fever spirochete Borrelia turicatae, early invasion of the brain occurs. Serotypes A and B are further distinguished by the abundant surface protein they produce: VmpA and VmpB, respectively. Western blotting with monoclonal antibodies, one-dimensional peptide mapping, and partial amino acid sequencing demonstrated regions of the VmpA protein that differed from VmpB. Oligonucleotide primers based on the partial amino acid sequences of unique regions were used to amplify a portion of the VmpA gene (vmpA) by PCR, and the product was used as a probe in Southern blot and Northern blot analyses. These experiments showed that (i) expression of the vmpA sequence was determined at the level of transcription and (ii) the vmpA sequence was in two locations in serotype A and one location in serotype B. The vmpA gene at the expression-linked locus of serotype A was cloned and sequenced. An open reading frame would encode a polypeptide of 214 amino acids. The polypeptide expressed by Escherichia coli was bound by VmA-specific but not VmpB-specific antibody. Primer extension analysis identified a consensus sigma70-type promoter for vmpA at the expression locus. Phylogenetic analysis revealed that VmpA is homologous to small Vmp (Vsp) proteins of B. hermsii and to OspC proteins of B. burgdorferi. These findings indicate that a function of the Vsp-OspC family of proteins of Borrelia spp. may be differential localization in organs, including the brain, during infection.

Publication Types:
PMID: 9234797 [PubMed - indexed for MEDLINE]

PMCID: PMC175474


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Ability of the Lyme disease spirochete Borrelia burgdorferi to infect rodents and three species of human-biting ticks (blacklegged tick, American dog tick, lone star tick) (Acari:Ixodidae).

Piesman J, Happ CM.

Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80522, USA.

The infectivity of a diverse collection of Borrelia burgdorferi strains from North America for mice was determined as a prelude to vector competence experiments with the 3 primary human-biting tick species in the eastern United States (Ixodes scapularis Say, Dermacentor variabilis (Say), Amblyomma americanum (L.)]. Of the 34 B. burgdorferi strains inoculated into mice, 29 were infectious; the exceptions were 5 isolates from Texas. Vector competence experiments were conducted with 2 strains from the southern United States (North Carolina and Georgia). Both strains were extremely infectious to I. scapularis larvae. Moreover, I. scapularis efficiently maintained these spirochetes transstadially and transmitted infection as nymphs. D. variabilis larvae were intermediate in susceptibility but generally did not maintain the infection transstadially. A. americanum larvae were completely refractory to infection with these 2 southern B. burgdorferi strains. Three isolates from Michigan D. variabilis were inoculated into mice, subsequently exposed to I. scapularis and D. variabilis larvae. Larval I. scapularis were 5-fold more susceptible to infection with these strains than were larval D. variabilis. Although nymphal I. scapularis efficiently transmitted a Michigan isolate, nymphal D. variabilis did not. In all these experiments, I. scapularis was the only species that proved to be vector competent for B. burgdorferi.

Publication Types:
PMID: 9220680 [PubMed - indexed for MEDLINE]

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Erratum in:
  • Cell 1999 Feb 5;96(3):447.

Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes.

Zhang JR, Hardham JM, Barbour AG, Norris SJ.

Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston 77030, USA.

We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.

Publication Types:
PMID: 9108482 [PubMed - indexed for MEDLINE]

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Oral delivery of purified lipoprotein OspA protects mice from systemic infection with Borrelia burgdorferi.

Luke CJ, Huebner RC, Kasmiersky V, Barbour AG.

Department of Microbiology, University of Texas Health Science Center at San Antonio 78284, USA.

The lipoprotein outer surface protein A (OspA) of the Lyme disease agent. Borrelia burgdorferi, has provided protection to mice and other animals against systemic infection when delivered orally as a recombinant protein in Escherichia coli, bacille Calmette. Guerin or Salmonella typhimurium. In the present study purified recombinant strain B31 OspA or outer surface protein D (OspD), another lipoprotein of B. burgdorferi, were administered either subcutaneously (s.c.) or orally without cell carrier or adjuvant to mice. In comparison to the OspD preparation, the OspA protein was 256-fold more resistant to trypsin. Whereas OspA in the suspension was in regular complexes of 17-25 nm in size, OspD formed amorphous globules of different sizes. Animals received a primary immunization and at least one booster. Mice immunized s.c. with either OspA or OspD had detectable antibodies to B. burgdorferi by enzyme-linked immunosorbent assay (ELISA), growth inhibition assay (GIA) and immunoblot. Delivered orally, OspA but not OspD elicited a specific antibody response, including IgA, as determined by these assays. The geometric mean titre of sera from mice who received 4 micrograms of OspA orally on days 1, 2, 4, 21 and 22 was 1470 by Ig ELISA, 320 by IgA ELISA and 128 by GIA. In infectious challenge experiments with B. burgdorferi strain Sh2-2-82 (OspA+ OspD- ) inoculated intradermally at 100 x the ID 50 all eight mice immunized with the 4 micrograms dose of OspA were protected, none of the mice immunized with the 4 micrograms dose of OspD were protected (P < 0.001 by Fisher exact test). These studies indicate that the lipoprotein OspA provides protection against systemic B. burgdorferi infection when delivered orally as a purified protein.

Publication Types:
PMID: 9178476 [PubMed - indexed for MEDLINE]

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Molecular characterization of a 6.6-kilodalton Borrelia burgdorferi outer membrane-associated lipoprotein (lp6.6) which appears to be downregulated during mammalian infection.

Lahdenne P, Porcella SF, Hagman KE, Akins DR, Popova TG, Cox DL, Katona LI, Radolf JD, Norgard MV.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75235, USA.

Isolated outer membranes of Borrelia burgdorferi 297 were utilized to obtain partial amino acid sequence information for a low-molecular-weight, outer membrane-associated polypeptide. Degenerate oligonucleotide primers based upon this information were used to amplify a 100-bp probe for detection of the corresponding full-length gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 17-amino-acid putative signal peptide terminated by LFVAC, a probable consensus sequence for lipoprotein modification, and a mature protein of 51 amino acids (predicted molecular mass of 5.8 kDa). The DNA sequences of the corresponding ORFs in B. burgdorferi 297 and B31 were identical; the corresponding ORF in strain N40 differed by only one nucleotide. Assuming conventional processing and acylation, the molecular weight of the lipoprotein, designated lp6.6, is about 6,600. The lp6.6 gene, which was localized to the 49-kb linear plasmid of B. burgdorferi, subsequently was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Immunoblot analysis with monoclonal antibody 240.7 revealed that lp6.6 was identical to a low-molecular-weight, highly conserved B. burgdorferi lipoprotein reported previously (L. I. Katona, G. Beck, and G. S. Habicht, Infect. Immun. 60:4995-5003, 1992). Results of indirect immunofluorescence assays, growth inhibition assays, passive immunizations, and active immunizations indicated that this outer membrane-associated antigen is not surface exposed in B. burgdorferi. Particularly interesting was the finding that mice and rhesus monkeys chronically infected with B. burgdorferi failed to develop antibodies against this antigen. We propose that high-level expression of lp6.6 is associated with the arthropod phase of the spirochetal life cycle and that expression of the gene is downregulated during mammalian infection.

Publication Types:
PMID: 9009290 [PubMed - indexed for MEDLINE]

PMCID: PMC174610


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An OspA-based DNA vaccine protects mice against infection with Borrelia burgdorferi.

Luke CJ, Carner K, Liang X, Barbour AG.

Department of Microbiology, University of Texas Health Science Center at San Antonio, USA.

Immunization with recombinant OspA protein of Borrelia burgdorferi protects against experimental Lyme disease. In the present study, mice were injected intramuscularly with plasmid DNA (VR2210) encoding strain B31 OspA. In this vector, the ospA-coding sequence was under transcriptional control of the cytomegalovirus immediate early promoter. For negative and positive controls, mice were immunized with either the plasmid vector without an osp-coding sequence or recombinant OspA protein, respectively. Mice immunized with VR2210 DNA produced OspA-specific antibodies that bound to B. burgdorferi in a whole cell ELISA and inhibited the growth of a homologous strain of B. burgdorferi. Immunization with VR2210 protected mice against challenge with 2 infectious strains of B. burgdorferi, Sh-2-82 and N40. These results indicate that vaccination with plasmid DNA expressing OspA is an efficacious method for providing a protective response against B. burgdorferi infection.

Publication Types:
PMID: 8985201 [PubMed - indexed for MEDLINE]

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Arthritis severity and spirochete burden are determined by serotype in the Borrelia turicatae-mouse model of Lyme disease.

Pennington PM, Allred CD, West CS, Alvarez R, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284, USA.

Immunodeficient mice infected with Borrelia turicatae, a relapsing fever agent, have a disorder that resembles disseminated Lyme disease. Two serotypes, A and B, differed in their arthritogenicity in both CB-17 SCID and C3H SCID mice. In CB-17 SCID mice infected with serotype A or B, arthritis was assessed by measurement of tibiotarsal diameter, functional ability on a beam walk test, and microscopic assessment of joint inflammation. Serotype B-infected mice had greater joint swelling, functional disability, and leukocytic infiltration in the joints than serotype A-infected mice. Joint swelling and disability peaked at 2 weeks of infection and then decreased, while leukocyte infiltration in the joints persisted. To investigate the basis for the differences in arthritogenicity of serotypes A and B, spirochete burdens in infected mice were measured by quantitative PCR of spirochete DNA in joints, direct immunofluorescence of spirochetes in joints, and counts of spirochetes in the blood. At 2 weeks of infection there were seven times more spirochetes in the joints of serotype B-infected mice than in those of serotype A-infected mice, measured by both quantitative PCR and direct enumeration. Although serotypes A and B had the same infectivity and growth rate in vivo, serotype B spirochetes were eightfold more abundant in the blood than serotype A spirochetes and produced greater fatality in newborn mice. These findings indicate that differences in disease severity in mice infected with serotype A or B are attributable to differences in the spirochete burden in the joints and blood.

Publication Types:
PMID: 8975925 [PubMed - indexed for MEDLINE]

PMCID: PMC174589


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Lyme borreliosis in the southern United States: a review.

Oliver JH Jr.

Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro 30460-8056, USA.

Lyme borreliosis (Lyme disease) is the most often reported arthropod transmitted disease in humans in the U.S.A. Although it has been reported from 43 states, cases are especially abundant in the mid-Atlantic and northeastern regions. Borrelia burgdorferi, the etiologic agent, is transmitted primarily by the western blacklegged tick (Ixodes pacificus) in far western North America, and by the blacklegged tick (Ixodes scapularis) in eastern North America. Although Lyme disease cases have been reported from southern states, some researchers doubt the presence of B. burgdorferi or of human Lyme disease in the south. However, new data show that B. burgdorferi is widely distributed in the south and that strains are genetically more varied than in the north. Moreover, B. burgdorferi enzootic cycles appear to be more complex and more tick species are identified as vectors of the spirochete in the southern states.

Publication Types:
PMID: 8973401 [PubMed - indexed for MEDLINE]

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The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.

Barbour AG, Carter CJ, Bundoc V, Hinnebusch J.

Department of Microbiology, University of Texas Health Science Center at San Antonio, 78284, USA. abarbour@uci.edu

A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O. The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B. burgdorferi. The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria.

Publication Types:
PMID: 8932323 [PubMed - indexed for MEDLINE]

PMCID: PMC178553


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In vivo activities of ceftriaxone and vancomycin against Borrelia spp. in the mouse brain and other sites.

Kazragis RJ, Dever LL, Jorgensen JH, Barbour AG.

Department of Medicine (Infectious Diseases), University of Texas Health Science Center at San Antonio 78284, USA.

Borrelia burgdorferi, the agent of Lyme disease, and B. turicatae, a neurotropic agent of relapsing fever, are susceptible to vancomycin in vitro, with an MIC of 0.5 microgram/ml. To determine the activity of vancomycin in vivo, particularly in the brain, we infected adult immunocompetent BALB/c and immunodeficient CB-17 scid mice with B. burgdorferi or B. turicatae. The mice were then treated with vancomycin, ceftriaxone as a positive control, or normal saline as a negative control. The effectiveness of treatment was assessed by cultures of blood and brain and other tissues. Ceftriaxone at a dose of 25 mg/kg of body weight administered every 12 h for 7 to 10 days eliminated cultivable B. burgdorferi or B. turicatae from all BALB/c or scid mice in the study. Vancomycin at 30 mg/kg administered every 12 h was effective in eliminating infection from immunodeficient mice if treatment was started within 3 days of the onset of infection. If treatment with vancomycin was delayed for 7 days or more, vancomycin failed to eradicate infection with B. burgdorferi or B. turicatae from immunodeficient mice. The failure of vancomycin in eradicating established infections in immunodeficient mice was associated with the persistence of viable spirochetes in the brain during antibiotic treatment.

Publication Types:
PMID: 8913478 [PubMed - indexed for MEDLINE]

PMCID: PMC163589


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Antibody responses of rats and humans to flagella-less cells and OspA protein of Borrelia burgdorferi.

Sadziene A, Thompson PA, Barbour AG.

Department of Microbiology, University of Texas Health Science Center at San Antonio 78284, USA.

Publication Types:
PMID: 8993358 [PubMed - indexed for MEDLINE]

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Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B.

Norgard MV, Arndt LL, Akins DR, Curetty LL, Harrich DA, Radolf JD.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235, USA.

There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease. A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide (LPS)- and lipoprotein-lipopeptide-induced immune cell signaling events. Like LPS, purified native B. burgdorferi OspA and synthetic analogs of OspA, OspB, and two T. pallidum lipoproteins (Tpp47 and Tpp17) all induced NF-kappa B translocation in THP-1 human monocytoid cells. Acylation of OspA and the synthetic peptides was requisite for cell activation. Polymyxin B abrogated only the response to LPS. By using 70Z/3-derived pre-B-cell lines either lacking or expressing human CD14 (the LPS receptor), it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides (assessed by induction of NF-kappa B and expression of surface immunoglobulin M). Finally, the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission. The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses.

Publication Types:
PMID: 8751937 [PubMed - indexed for MEDLINE]

PMCID: PMC174301


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Parasites and selected diseases of collared peccaries (Tayassu tajacu) in the trans-pecos region of Texas.

Gruver KS, Guthrie JW.

Denver Wildlife Research Center, United States Department of Agriculture, Colorado 80225-0266, USA.

Fifty-five collared peccaries (Tayassu tajacu) were collected from October 1988 through April 1991 from five counties within the Trans-Pecos region of Texas (USA) to monitor for diseases and parasites. No endoparasites were recovered on gross examination. Antibody to Borrelia burgdorferi was documented in one (2%) of 55 specimens. Three (6%) of 54 collared peccaries were positive for Yersinia pestis antibodies. All collared peccaries were negative for antibodies against Brucella spp., Francisella tularensis, Rickettsia rickettsii, and Rickettsia typhi. This is the first report of Borrelia sp. and Yersinia sp. pathogens in collared peccaries from this region of Texas.

Publication Types:
PMID: 8827690 [PubMed - indexed for MEDLINE]

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Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.

Porcella SF, Popova TG, Akins DR, Li M, Radolf JD, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, 75235, USA.

DNA sequencing and Southern blot analyses of a Borrelia burgdorferi DNA fragment encoding a signal sequence led to the discovery of a genetic locus, designated 2.9, which appears to be present in at least seven copies in virulent B. burgdorferi 297. DNA sequence analysis of these regions revealed that each 2.9 locus contained an operon of four genes (ABCD) and open reading frames designated rep+ (positive strand) and rep- (negative strand) which encoded multiple repeat motifs. Downstream of the rep+ gene(s) in six of the completely cloned and sequenced 2.9 loci also were lipoprotein (LP) genes possessing highly similar signal sequences but encoding variable mature polypeptides. The lipoproteins could he separated into two classes on the basis of hydrophilicity profiles, sequence similarities, and reactivity with specific antibodies. The 2.9 loci were localized to two (20- and 30-kb) supercoiled plasmids in B. burgdorferi 297. Northern (RNA) blot analysis established that the 2.9 ABCD operon was only minimally expressed, whereas the rep- gene(s) and at least three of the seven LP genes were expressed by B. burgdorferi in vitro. A single putative promoter element was identified by RNA primer extension analysis upstream of the ABCD operon, whereas a number of potential promoter regions existed upstream of the LP genes. The combined data indicate that the ABCD operon, rep+ and rep- genes, and LP genes are separately transcribed during in vitro growth. The 2.9 loci possess a repetitiveness, diversity, and complexity not previously described for B. burgdorferi; differential expression of these genes may facilitate the spirochete's ability to survive in diverse host environments.

Publication Types:
PMID: 8655511 [PubMed - indexed for MEDLINE]

PMCID: PMC178083


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A flagella-less mutant of Borrelia burgdorferi as a live attenuated vaccine in the murine model of Lyme disease.

Sadziene A, Thompson PA, Barbour AG.

Department of Medicine (Infectious Diseases), University of Texas Health Science Center at San Antonio 78284, USA.

The immunogenicity and protective efficacy of an attenuated isolate of Borrelia burgdorferi, the agent of Lyme disease, were evaluated. An isogenic pair of isolates of strain HB19 differed in the expression of flagella; neither produced systemic infection or persisted in the skin of mice. In a comparison of intradermally administered live flagella-bearing and flagella-less cells, the flagella-less isolate was equal to or superior to the flagella-bearing wild type in eliciting growth-inhibiting antibodies and preventing infection in mice. In a comparison of live and killed flagella-less cells, immune responses to live cells were superior to those to killed cells, as assessed by ELISA, growth inhibition assay, and infectious challenge. The dose protecting 50% of mice was 10(3.8) and 10(5.2) for live and killed cells, respectively (P < .05).

Publication Types:
PMID: 8627071 [PubMed - indexed for MEDLINE]

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Experimental immunization against Lyme borreliosis with recombinant Osp proteins: an overview.

Sadziene A, Barbour AG.

Dept. of Microbiology and Medicine, University of Texas Health Science Center at San Antonio 78284-7758, USA.

Interest in human and veterinary vaccines against Lyme borreliosis is growing. Both whole cell immunization and subunit vaccines can protect against infection with Borrelia burgdorferi. For development of a human vaccine the focus has been on a subunit vaccine. The most promising candidate is OspA, a major outer membrane lipoprotein of B. burgdorferi sensu lato. Of Osp proteins A through D, OspA shows the least variability between strains in its sequence and in the level of its expression. Borreliae in ticks express OspA. Antibodies to OspA kill borreliae in vitro and provide passive protection in mice. Active immunization of mice with OspA provides protection against challenge by syringe inoculation or tick bite. The lipid moiety of the OspA is necessary for immunogenicity in the absence of a potent adjuvant. A recombinant OspA-based vaccine is already in clinical trials. Although there is compelling evidence that immunization with OspA will provide protection, questions remain regarding the duration of protection from such immunization, the necessity to have a minimum level of neutralizing antibodies at all times for protection, and the relationship of an immune response to OspA and autoimmune features of Lyme borreliosis. The experimental aspects of immunization with Osp-A based constructs and other Lyme vaccine candidates are reviewed and discussed.

Publication Types:
PMID: 8740122 [PubMed - indexed for MEDLINE]

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Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness.

Barbour AG, Maupin GO, Teltow GJ, Carter CJ, Piesman J.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284, USA.

Bites from the hard tick Amblyomma americanum are associated with a Lyme disease-like illness in the southern United States. To identify possible etiologic agents for this disorder, A. americanum ticks were collected in Missouri, Texas, New Jersey, and New York and examined microscopically. Uncultivable spirochetes were present in approximately 2% of the ticks. Borrelia genus-specific oligonucleotides for the flagellin and 16S rRNA genes were used for amplification of DNA. Products were obtained from ticks containing spirochetes by microscopy but not from spirochete-negative ticks. Sequences of partial genes from spirochetes in Texas and New Jersey ticks differed by only 2 of 641 nucleotides for flagellin and 2 of 1336 nucleotides for 16S rRNA. Phylogenetic analysis showed that the spirochete was a Borrelia species distinct from previously characterized members of this genus, including Borrelia burgdorferi. Gene amplification could be used to detect these spirochetes in ticks and possible mammalian hosts.

Publication Types:
PMID: 8568302 [PubMed - indexed for MEDLINE]

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Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.

Ferdows MS, Serwer P, Griess GA, Norris SJ, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284, USA.

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.

Publication Types:
PMID: 8550515 [PubMed - indexed for MEDLINE]

PMCID: PMC177727


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Does Lyme disease occur in the south?: a survey of emerging tick-borne infections in the region.

Barbour AG.

Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284, USA.

Lyme disease is the most common arthropod-borne infection in the United States. However, the risk of infection varies widely by geographic region. In the South, Borrelia burgdorferi has been identified in ticks and small mammals, but transmission of the agent to humans has not been documented. The Lyme disease-like disorder reported from the region may have another etiology.

Publication Types:
PMID: 8571985 [PubMed - indexed for MEDLINE]

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Borrelia burgdorferi vesicle production occurs via a mechanism independent of immunoglobulin M involvement.

Shoberg RJ, Thomas DD.

Department of Periodontics, University of Texas Health Science Center at San Antonio 78284-7894, USA.

Borrelia burgdorferi produces extracellular vesicles containing various borrelial protein antigens when propagated in vitro in culture media. Commonly observed components of borrelial vesicle preparations are borrelial surface antigens, bovine serum albumin, and the heavy chains of rabbit immunoglobulin G and immunoglobulin M. This study employed ultracentrifugation to harvest borrelial vesicles and analyzed these preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. We demonstrated that the rabbit mu heavy-chain band observed was devoid of OspA or at most levels below those detectable by immunoblot. We also demonstrated the recovery of borrelial vesicles at relative centrifugal forces as low as 25,000 x g, compared with the force of > 200,000 x g normally employed. Further, the mu heavy-chain band was recovered from uninoculated growth media processed at 25,000 x g, suggesting that it behaves as a particle rather than as a soluble molecule under these conditions. Lastly, vesicles were demonstrated to be present in preparations harvested from growth media supplemented with fetal calf serum, suggesting that vesicle production by B. burgdorferi can occur in the absence of immunoglobulins.

Publication Types:
PMID: 7591146 [PubMed - indexed for MEDLINE]

PMCID: PMC173695


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Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue.

Akins DR, Porcella SF, Popova TG, Shevchenko D, Baker SI, Li M, Norgard MV, Radolf JD.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.

Protein export signals from the low-passage 297 strain of Borrelia burgdorferi were cloned as fusions with an Escherichia coli alkaline phosphatase (PhoA) reporter lacking a signal sequence. One PhoA+ clone (BbK2.10-PhoA) was derived from a borrelial lipoprotein. Although the polypeptide encoded by the full-length bbk2.10 gene had 76% similarity and 56% identity to outer surface protein F (OspF) from B. burgdorferi strain N40, antibodies directed against recombinant forms of the two proteins revealed that they were not cross-reactive. The nucleotide sequences of bbk2.10 and ospF from the N40 and 297 strains, respectively, were determined to confirm that the N40 and 297 strains each contained both genes. Southern blot analysis revealed that bbk2.10 is a single-copy gene and that the B. burgdorferi strain 297 and N40 genomes appeared to contain one other gene more closely related to ospF than bbk2.10. It was particularly noteworthy that ospF, but not bbk2.10, was expressed in vitro while B. burgdorferi-infected mice generated antibodies reactive with both lipoproteins. To help confirm that the BbK2.10-reactive antibodies produced by the B. burgdorferi-infected mice were specific for that protein, a second gene, bbk2.11, which hybridized with the ospF probe was cloned; the corresponding polypeptide reacted strongly with OspF antisera but failed to react with BbK2.10-specific antisera. Taken together, these data demonstrate that BbK2.10, BbK2.11, and OspF comprise a B. burgdorferi lipoprotein family and that at least one member (BbK2.10) appears to be expressed only during infection.

Publication Types:
PMID: 8748034 [PubMed - indexed for MEDLINE]

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Modulation of immunity to Borrelia burgdorferi by ultraviolet irradiation: differential effect on Th1 and Th2 immune responses.

Brown EL, Rivas JM, Ullrich SE, Young CR, Norris SJ, Kripke ML.

Department of Immunology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.

Ultraviolet B (UVB) radiation suppresses the delayed-type hypersensitivity (DTH) response to alloantigen by a mechanism involving interleukin (IL)-10. It has been hypothesized, based on this result, that UV irradiation shifts the immune response from a Th1 to a Th2 response. We tested this hypothesis using Borrelia burgdorferi (Bb) as an antigen under conditions where both DTH and antibody responses could be assessed. Mice were irradiated with a single dose of UV and then immunized with Bb in complete Freund's adjuvant (CFA). DTH was assessed by footpad challenge. At various time points thereafter, mice were bled, and the serum antibodies to Bb were quantitated. Only IgG1, IgG2a, and IgG2b were produced in response to Bb. The IgG2a and IgG2b antibody responses, as well as the DTH response to Bb, showed UV dose-dependent reductions after UV irradiation. The primary IgG1 response to Bb was very low and was unaffected by UV irradiation; however, the IgG1 secondary response was elevated in UV-irradiated mice. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the IgG2a and IgG2b antibody responses. In addition, injecting recombinant murine IL-10 mimicked some of the effects of UV radiation. Our results support the hypothesis that in vivo, UV irradiation down-regulates Th1 immune responses, while leaving Th2 responses intact, and suggest that IL-10 is an important mediator of this effect.

Publication Types:
PMID: 7489737 [PubMed - indexed for MEDLINE]

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Chemiluminescent analysis of Borrelia burgdorferi penicillin-binding proteins using ampicillin conjugated to digoxigenin.

Norgard MV, Baker SI, Radolf JD.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235, USA.

Knowledge of the penicillin-binding proteins (PBPs) of Borrelia burgdorferi is important for understanding both the targets of beta-lactams used therapeutically for Lyme borreliosis and the complex membrane biology of the distinctive spirochetal pathogen which causes Lyme disease. In this study, the PBPs of a number of B. burgdorferi strains and variants were examined using a rapid and sensitive chemiluminescent assay which employs ampicillin conjugated to digoxigenin (dig-amp). The minimum inhibitory concentration of dig-amp for B. burgdorferi high-passage strain B31 (0.012 micrograms/ml) was essentially no different from that of free ampicillin (0.025 micrograms/ml). Dig-amp bound specifically to B. burgdorferi B31 PBPs with molecular masses of 92, 80, 65, 46, 40, 34, 31, 29, 22, 20 and 13 kDa; the 31 kDa and 34 kDa PBPs were proven to be OspA and OspB, respectively. All of the borrelial PBPs were present in the cytoplasmic membrane fraction of B. burgdorferi, findings consistent with their activities as PBPs but inconsistent with OspA and OspB as surface-exposed outer membrane lipoproteins. Furthermore, among the PBP profiles of other high- and low-passage variants of B. burgdorferi strains Sh-2-82, HB19, and N40, which differed somewhat from one another, OspD (28 kDa) but not OspC (22-25 kDa) also was strongly implicated as a PBP; however, OspC possessed a gel mobility easily misconstrued as that of a 26 kDa PBP often expressed reciprocally with OspB. The ramifications of classifying OspA, OspB, and OspD as PBPs are discussed. While the current inability to genetically manipulate B. burgdorferi hinders determining which of the borrelial PBPs are essential for spirochetal viability (i.e., are the lethal targets of beta-lactams), a priori knowledge of the borrelial PBPs will facilitate the production and purification of recombinant derivatives whose activities can be assessed further in vitro.

Publication Types:
PMID: 8825913 [PubMed - indexed for MEDLINE]

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Adherence of Borrelia burgdorferi to the proteoglycan decorin.

Guo BP, Norris SJ, Rosenberg LC, Höök M.

Department of Biochemistry and Biophysics, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University, Houston 77030, USA.

Lyme disease is a tick-borne infection that can develop into a chronic, multisystemic disorder. The causative agent, Borrelia burgdorferi, is initially deposited by the tick into the host dermis, where it associates with collagen fibers, replicates, and eventually disseminates to other tissues. We have examined the adherence of the spirochete to different components of the collagen fiber and demonstrated that decorin, a proteoglycan which decorates collagen fibers, can support the attachment of B. burgdorferi. No significant direct attachment to isolated type I or III collagens could be detected. Attachment of the spirochetes to decorin was highly specific, and the process could be inhibited by soluble decorin but not by various unlabeled, unrelated components. B. burgdorferi also bound soluble 125I-labeled decorin in a time- and concentration-dependent manner. Spirochete binding of soluble 125I-labeled decorin required intact proteoglycan and could not be inhibited by either isolated core protein or glycosaminoglycan chain. B. burgdorferi expresses two decorin-binding proteins with apparent molecular masses of 19 and 20 kDa as revealed in a Western blot (immunoblot)-type assay. Our results indicate that decorin may mediate the adherence of B. burgdorferi to collagen fibers in skin and other tissues.

Publication Types:
PMID: 7642279 [PubMed - indexed for MEDLINE]

PMCID: PMC173478


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Membrane topology of Borrelia burgdorferi and Treponema pallidum lipoproteins.

Jones JD, Bourell KW, Norgard MV, Radolf JD.

Department of Microbiology, University of Texas, Southwestern Medical Center at Dallas 75235, USA.

A critical issue regarding the molecular architectures of Treponema pallidum and Borrelia burgdorferi, the agents of venereal syphilis and Lyme disease, respectively, concerns the membrane topologies of their major lipoprotein immunogens. A related question is whether these lipid-modified membrane proteins form intramembranous particles during freeze fracture electron microscopy. To address these issues, native borrelial and treponemal lipoproteins were reconstituted into liposomes of diverse composition. The importance of the covalently associated lipids for membrane association of lipoproteins was revealed by the observation that nonlipidated recombinant forms of both B. burgdorferi OspA and the T. pallidum 47-kDa immunogen (Tpp47) showed very weak or no binding to model bilayer vesicles. In contrast to control liposomes reconstituted with bacteriorhodopsin or bovine rhodopsin, two well-characterized transmembrane proteins, none of the lipoprotein-liposomes contained particles when examined by freeze fracture electron microscopy. To extend these findings to prokaryotic lipoproteins with relatively amphiphilic polypeptides, similar experiments were conducted with a recombinant nonlipidated form of Escherichia coli TraT, a lipoprotein which has putative transmembrane domains. The nonlipidated TraT oligomers bound vesicles derived from E. coli lipids but, surprisingly, did not form particles in the freeze-fractured liposomes. These findings support (i) a proposed topology of spirochetal lipoproteins in which the polypeptide is extrinsic to the membrane surface and (ii) the contention that particles visualized in freeze-fractured spirochetal membranes represent poorly characterized transmembrane proteins.

Publication Types:
PMID: 7790053 [PubMed - indexed for MEDLINE]

PMCID: PMC173324


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High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi.

Norris SJ, Howell JK, Garza SA, Ferdows MS, Barbour AG.

Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston 77225, USA.

Borrelias that cause Lyme disease lose the ability to infect and cause disease in laboratory animals following 10 to 16 passages of in vitro culture. In this study, clonal populations of the Sh-2-82 (Sh2) and B31 strains of Borrelia burgdorferi were isolated by subsurface plating on BSK-II agar plates and examined for infectivity in the C3H/HeN mouse model. Mice were injected intradermally with 10(5) B. burgdorferi organisms, and the tibiotarsal joint, heart, and bladder were cultured 2 to 4 weeks postinfection to determine whether viable organisms were present. Clones exhibited either a high-infectivity phenotype, in which cultures were consistently positive at all organ sites, or a low-infectivity phenotype, in which a low proportion of cultures were positive (5 of 40 in a representative experiment). In an Sh2 population that had undergone five in vitro passages, 7 of 10 clones were of the high-infectivity phenotype, and the remaining clones were of the low-infectivity phenotype. The proportion of high-infectivity clones decreased with continued in vitro passage, with only 1 of 10 clones exhibiting the high-infectivity phenotype after 10 passages and 0 of 10 clones yielding positive cultures after 20 passages. Representative high- and low-infectivity clones from passage 5 Sh2 cultures had 50% infectious doses of 1.8 x 10(2) and 1 x 10(5), respectively. Subclones consistently reflected the same infectivity phenotypes as those of the parent clones. The protein profiles and plasmid contents of the high- and low-infectivity clones were compared and exhibited few discernible differences. On the basis of these results, the loss of infectivity during in vitro culture results from the outgrowth of low-infectivity clones and begins to occur within the first five in vitro passages. Further examination of clonal populations may lead to the identification of genetic and protein factors important in the virulence and pathogenicity of Lyme disease borrelias.

Publication Types:
PMID: 7768600 [PubMed - indexed for MEDLINE]

PMCID: PMC173287


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Borrelia burgdorferi mutant lacking Osp: biological and immunological characterization.

Sadziene A, Thomas DD, Barbour AG.

Department of Microbiology and Medicine, University of Texas Health Science Center at San Antonio 78284.

All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of B. burgdorferi lacking Osps were selected with polyclonal or monoclonal antibodies at a frequency of 10(-6) to 10(-5). One mutant that lacked OspA, -B, -C, and -D was further characterized. It was distinguished from the OspA+B+ cells by its (i) autoaggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum and complement sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in mouse skin for the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two types of monoclonal antibodies (MAbs) with growth-inhibiting properties for mutant cells were identified. The first type bound to a 13-kDa surface protein of B. burgdorferi sensu stricto and of B. afzelii. The MIC of the Fab fragment of one MAb of this type was 0.2 micrograms/ml. The second type of MAb to the Osp-less mutant did not bind to B. burgdorferi components by Western blotting (immunoblotting) but did not bind to unfixed, viable cells in immunofluorescence and growth inhibition assays. These studies revealed possible functions Osp proteins in borrelias, specifically serum resistance, and indicated that in the absence of Osp proteins, other antigens are expressed or become accessible at the cell surface.

Publication Types:
PMID: 7890424 [PubMed - indexed for MEDLINE]

PMCID: PMC173191


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Dermal inflammation elicited by synthetic analogs of Treponema pallidum and Borrelia burgdorferi lipoproteins.

Norgard MV, Riley BS, Richardson JA, Radolf JD.

Department of Microbiology, University of Texas Southwestern Medical Center at Dallas 75235.

The membrane lipoproteins of Treponema pallidum and Borrelia burgdorferi have potent immunostimulatory properties in vitro, implicating them as major inflammatory mediators in syphilis and Lyme disease. Recently, we reported that synthetic lipohexapeptide analogs (lipopeptides) of the lipoproteins could be used as surrogates for native spirochetal lipoproteins in immune cell activation studies in vitro. The present study was designed to evaluate the inflammatory properties of the lipopeptides in vivo and to correlate the cellular responses to these synthetic analogs with the histopathology of syphilis and Lyme disease. Lipopeptides corresponding to the 47-kDa major membrane lipoprotein of T. pallidum and the outer surface protein A of B. burgdorferi injected intradermally induced dose-dependent dermal inflammation in mice; the initial predominantly neutrophilic (mice) or heterophilic (rabbits) cellular infiltrates were followed by infiltrates consisting predominantly of mononuclear cells. The intradermal response of rabbits to the 47-kDa lipopeptide was strikingly similar to that observed for animals infected intradermally with T. pallidum. In all cases, lipopolysaccharide was substantially more potent as an inflammatory mediator than the spirochetal lipopeptides. In contrast to the lipopeptides, nonacylated hexapeptides elicited minimal or no dermal lesions in mice or rabbits, underscoring the importance of acyl modification to the inflammatory properties of the lipopeptides. This study provides the first in vivo evidence that the spirochetal lipoproteins/lipopeptides contribute to the immunopathogenesis of syphilis and Lyme disease.

Publication Types:
PMID: 7890417 [PubMed - indexed for MEDLINE]

PMCID: PMC173182


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Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytes/macrophages.

Radolf JD, Arndt LL, Akins DR, Curetty LL, Levi ME, Shen Y, Davis LS, Norgard MV.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.

The observation that the major membrane immunogens of the spirochetal pathogens. Treponema pallidum and Borrelia burgdorferi are lipoproteins prompted studies to investigate macrophage activation by the 47-kDa lipoprotein of T. pallidum and the acylated outer surface protein A (OspA) of B. burgdorferi. Both lipoproteins induced the synthesis of biologically active TNF-alpha and chloramphenicol acetyltransferase in a murine macrophage cell line transfected with a chloramphenicol acetyltransferase reporter gene controlled by a TNF promoter (TB2 cells). Nonacylated forms of these polypeptides did not induce cell activation. Comparison between purified OspA and B. burgdorferi cellular lipids revealed that the former was the more potent inducer of TNF-alpha. Synthetic lipohexapeptides corresponding to the N-termini of the 47-kDa lipoprotein of T. pallidum and OspA also activated TB2 cells in a dose-dependent fashion, whereas the nonlipidated hexapeptides were without effect, further underscoring the importance of protein acylation to cell activation. Among several lines of evidence supporting that macrophage stimulation by LPS and lipopeptides proceeds via different mechanisms, the most notable was that lipopeptides activated peritoneal macrophages from LPS-nonresponsive C3H/HeJ mice. The potential for spirochetal lipoproteins to function as general macrophage activators was demonstrated by the ability of the synthetic analogues to induce IL-1 beta, IL-6, and IL-12, in addition to TNF, in murine and/or human macrophages. Our findings indicate that spirochetal lipoproteins may be important immunomodulators in syphilis and Lyme disease and that the synthetic lipopeptides will be useful surrogates for studying immune mechanisms operative in the two spirochetal diseases.

Publication Types:
PMID: 7876555 [PubMed - indexed for MEDLINE]

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Experimental feline Lyme borreliosis as a model for testing Borrelia burgdorferi vaccines.

Gibson MD, Omran MT, Young CR.

Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station 77843-4458, USA.

The feline model investigated establishes that domestic cats may act as an animal model for evaluating the pathogenesis of Lyme borreliosis. Specifically this feline model demonstrates: First, that animals seroconvert following either needle injection of, or arthropod delivery of, Borrelia burgdorferi. Clinical findings obtained are consistent with those observed in human Lyme disease; histopathological observations are also consistent with those observed in human Lyme disease. Therefore, cats may also be used as a representative animal model for measuring immune protection against Lyme borreliosis. Specifically we are exploring the protective capacity of Borrelia burgdorferi antigenic compounds in cats, namely OspA, OspB, OspC, heat shock proteins, flagellar antigens and various protective immunological combinations.

Publication Types:
PMID: 8644516 [PubMed - indexed for MEDLINE]

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[Growth inhibition of Borrelia burgdorferi sensu lato by antibodies. A contribution to understanding the pathogenesis and improving diagnosis of Lyme borreliosis]

[Article in German]

Sadziene A, Barbour AG.

Abteilung für Mikrobiologie und Medizin, Universität von Texas, San Antonio, USA.

Phenomenon of growth inhibition of Borrelia burgdorferi sensu lato (BBSL), the agent of Lyme disease, by antiborrelial antibodies was observed and investigated. Some of the antiborrelial antibodies were bactericidal in the absence of complement and phagocytes. Based on growth inhibition phenomenon we developed and evaluated the function-oriented in vitro growth inhibition assay (GIA). GIA proved to be more strain-specific and a better predictor of protection against infectious challenge than matrix-based assays, such as ELISA and Western blot. Growth inhibition phenomenon was also applied as a tool for selection of antibody-resistant BBSL mutants. All BBSL isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of BBSL with altered or absent Osps were selected with polyclonal or monoclonal antibodies (mAbs) at a frequency of 10(-2) to 10(-6). Based on PAGE and Western blot analysis all examined mutants were catalogued into 4 classes. Some of these mutants were later employed in studies of functional importance of Osps in immunopathogenesis of BBSL. Several studies revealed some possible functions of Osp proteins in borrelias. In one study, Osp-lacking mutant was distinguished from its Osp-bearing parent by autoaggregation and slower growth rate, diminished capacity to adhere to human umbilical vein endothelial cells, decreased plating efficiency on solid medium as well as serum and complement sensitivity. Mutant also was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant cells survived in mouse skin for the same duration as wild type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types:
PMID: 7610664 [PubMed - indexed for MEDLINE]

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Role of outer membrane architecture in immune evasion by Treponema pallidum and Borrelia burgdorferi.

Radolf JD.

Dept of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas 75235.

Combined ultrastructural and molecular studies have revealed that the syphilis and Lyme-disease spirochetes, Treponema pallidum and Borrelia burgdorferi, have distinctive molecular architectures. Both organisms persist in their hosts and have strategies for immune evasion that include the use of rare, poorly immunogenic surface-exposed proteins as potential virulence determinants.

Publication Types:
PMID: 7812663 [PubMed - indexed for MEDLINE]

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Seroprotective groups of Lyme borreliosis spirochetes from North America and Europe.

Lovrich SD, Callister SM, Lim LC, DuChateau BK, Schell RF.

Wisconsin State Laboratory of Hygiene, University of Wisconsin, Madison.

Five distinct seroprotective groups among North American and European isolates of Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii have been identified using the in vitro borreliacidal assay. The predominant North American seroprotective group comprised isolate 297 and B. burgdorferi isolates from California, Illinois, Wisconsin, New York, and Texas. A second group was represented by isolate C-1-11. The majority of European isolates belonged to a seroprotective group composed of B. garinii. Another European group contained isolates classified genetically as genospecies group VS461 (B. afzelii). A fifth group, represented by isolate LV5, could kill both North American and European isolates of B. burgdorferi sensu lato. These results suggest that combinations of immunogenic protective proteins of spirochetes will be necessary to provide a comprehensive vaccine.

Publication Types:
PMID: 8014485 [PubMed - indexed for MEDLINE]

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A family of surface-exposed proteins of 20 kilodaltons in the genus Borrelia.

Carter CJ, Bergström S, Norris SJ, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.

Relapsing fever and Lyme disease spirochetes of the genus Borrelia display at their surfaces abundant lipoproteins: Vmp proteins in Borrelia hermsii and Osp proteins in Borrelia burgdorferi. Vmp and Osp proteins largely determine serotype specificity, and neutralizing antibodies of infected or immunized animals are directed at them. For the present study, we examined B. hermsii serotype 33, which is unique among strain HS1 serotypes in the low frequency of switches to other serotypes during infections and in vitro cultivation. Failing to clone the complete vmp33 gene, we accomplished its further characterization by (i) determining three partial amino acid sequences, (ii) designing oligonucleotide primers based on these amino acid sequences, (iii) cloning and sequencing the central portion of vmp33, and (iv) using outwardly directed primers and the inverse PCR to clone the 5' and 3' ends of the gene and flanking regions. The transcriptional start site was identified by primer extension analysis. Vmp33 was a polypeptide of 211 amino acids; the three partial amino acid sequences were identified in the open reading frame. Vmp33 was found to be more similar to other 20-kDa Vmp proteins of B. hermsii and to OspC proteins of B. burgdorferi than it was to 35- to 39-kDa Vmp proteins of the same strain. Moreover, OspC proteins were more similar to Vmp33 than they were to OspA, -B, or -D proteins of B. burgdorferi. These sequence similarities were consistent with Western blot (immunoblot) findings of cross-reactions between Vmp33 and OspC with anti-Vmp33 and anti-OspC sera. The promoter for the expressed vmp33 gene was found to be different from the expression site for other active vmp genes characterized to date. These results indicate that Vmp33 and other small Vmp's belong with OspC to a genus-wide family of 20-kDa proteins and that expression of these proteins may be coordinated with expression of other Vmp and Osp proteins in Borrelia spp.

Publication Types:
PMID: 8005669 [PubMed - indexed for MEDLINE]

PMCID: PMC302883


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A bactericidal antibody to Borrelia burgdorferi is directed against a variable region of the OspB protein.

Sadziene A, Jonsson M, Bergström S, Bright RK, Kennedy RC, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.

Borrelia burgdorferi, an agent of Lyme disease, is killed by some monoclonal antibodies in the absence of complement or phagocytes. In the present study, the bactericidal action of monoclonal antibodies against B. burgdorferi and B. hermsii, a cause of relapsing fever, was further characterized. H6831, an antibody recognizing the OspB proteins of some B. burgdorferi strains, and H4825, an antibody specific for one serotype of B. hermsii, were purified, and Fab fragments of the antibodies were prepared. In time-kill studies, more than 99.9% of strain B31 B. burgdorferi cells were killed after 30 min of exposure to H6831 Fab fragments. The MBC of the Fab fragments was 10 micrograms/ml. Electron microscopy revealed that the bactericidal Fab fragments produced numerous blebs and cell lysis of the borrelias for which they were specific. To identify the epitope for H6831, the OspB sequences of H6831-susceptible and -resistant strains and mutants were determined. The deduced OspB proteins of all H6831-resistant strains and mutants differed from the strain B31 OspB at residue 253. Murine antisera raised against a 21-mer synthetic peptide representing the region around residue 253 were specific for strain B31 by Western blot (immunoblot) and growth inhibition assays. Furthermore, the antipeptide serum inhibited the binding of H6831 to whole borrelias. These findings indicated that the linear component of the bactericidal antibody's epitope was located at or near residue 253.

Publication Types:
PMID: 7513309 [PubMed - indexed for MEDLINE]

PMCID: PMC186463


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Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

Belisle JT, Brandt ME, Radolf JD, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center at Dallas 75235.

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.

Publication Types:
PMID: 8157583 [PubMed - indexed for MEDLINE]

PMCID: PMC205333


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Solid-phase synthesis of biologically active lipopeptides as analogs for spirochetal lipoproteins.

DeOgny L, Pramanik BC, Arndt LL, Jones JD, Rush J, Slaughter CA, Radolf JD, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center at Dallas 75235.

Bacterial lipoproteins, which are of particular interest because of their immunomodulatory activities, share a common N-terminal structural motif that consists of an N-acyl-S-diacylglyceryl cysteine residue. Synthetic tripalmitoylated analogs of the N-terminal sequences of several bacterial lipopetides have been found to reproduce the immunological activities of the corresponding intact lipoproteins. Methods for the synthesis of lipopeptide analogs of bacterial lipoproteins have hitherto relied upon the coupling of peptide moieties, lacking the N-terminal cystienyl residue, with a tripalmitoylglyceryl cysteine moiety synthesized separately in solution. A method is described here by which rapid and convenient synthesis of the entire lipopeptide is accomplished by solid-phase methods in which the N-terminal cysteinyl derivative is assembled stepwise while attached to the completed peptide moiety prior to cleavage from the resin. The method has been used to synthesize two lipohexapeptides representing the N-terminal sequences of the 47-kDa membrane lipoprotein of the syphilis spirochete, Treponema pallidum, and the outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi. These lipopeptides, which were synthesized without detectable endotoxin contamination, exhibit macrophage-stimulating activity that is not expressed by the corresponding non-acylated hexapeptides. The data indicate that synthetic lipopeptides based on spirochetal lipoproteins are appropriate substitutes for the intact lipoproteins in immunological studies.

Publication Types:
PMID: 8012126 [PubMed - indexed for MEDLINE]

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Variability of a bacterial surface protein and disease expression in a possible mouse model of systemic Lyme borreliosis.

Cadavid D, Thomas DD, Crawley R, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

During persistent infection of scid mice with Borrelia turicatae, an agent of relapsing fever and neuroborreliosis, there was variation in the surface proteins the bacteria expressed and in disease manifestations over time. Two serotypes, A and B, were isolated from the mice, cloned by limiting dilution, and further characterized. The only discernible difference between the two variants was in the size of the major surface protein they expressed: serotype A had a variable major protein (Vmp) of 23,000, and serotype B had a Vmp of 20,000. When other scid mice were inoculated with clonal populations of A and B, the infections were similar with respect to onset and degree of spirochetemia, involvement of the eye and heart, and occurrence of a peripheral vestibular disorder. However, there were differences between the serotypes in other respects: (a) serotype B but not A caused reddened and significantly enlarged joints, markedly impaired performance on a walking bar, and severe arthritis by histologic examination; (b) serotype A but not B invaded the central nervous system during early infection; and (c) serotype A penetrated monolayers of human umbilical vein endothelial cells more readily than did serotype B. The combination of arthritis, myocarditis, and neurologic disease resembled human Lyme borreliosis. The findings indicate that differences in disease expression are determined by variable surface proteins of the bacterium and that scid mouse infections with B. turicatae provide a model for the study of the pathogenesis of Lyme borreliosis and other persistent spirochetal diseases.

Publication Types:
PMID: 8294872 [PubMed - indexed for MEDLINE]

PMCID: PMC2191368


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Differential association of Borrelia species with cultured neural cells.

Thomas DD, Cadavid D, Barbour AG.

Department of Periodontics, University of Texas Health Science Center at San Antonio 78284.

Studies of the interactions of relapsing fever Borrelia species with cultured neural cells have not been reported. In the present work, the interaction of the relapsing fever agents Borrelia hermsii and Borrelia turicatae with cultured neural and endothelial cells was studied and compared with Borrelia burgdorferi, the agent of Lyme disease. All Borrelia species bound each cell type tested. Host cell association was time-dependent and saturable. With the exception of human neuron cells, B. hermsii and B. turicatae associated to a greater degree with neural-derived cell types than did B. burgdorferi. In contrast, B. burgdorferi associated better with endothelial cells than did the relapsing fever borreliae. B. burgdorferi were found in human neuron cells to compete for association sites with B. hermsii and B. turicatae, suggesting the existence of some identity between the ligands or receptors used. Data indicate that the study of in vitro association of Borrelia species with neural-derived cells may provide valuable information for a better understanding of pathogenic mechanisms in neuroborreliosis.

Publication Types:
PMID: 8106781 [PubMed - indexed for MEDLINE]

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Identification of a highly cross-reactive outer surface protein B epitope among diverse geographic isolates of Borrelia spp. causing Lyme disease.

Shoberg RJ, Jonsson M, Sadziene A, Bergström S, Thomas DD.

Department of Periodontics, University of Texas Health Science Center at San Antonio 78284.

The outer surface lipoprotein B (OspB) of Borrelia burgdorferi is a major component of the borrelial protein profile and has been shown to be highly immunogenic in experimentally immunized and infected mammals. However, the ospB loci of different strains show considerable heterology at the nucleic acid sequence level, and the progeny of a clonal strain of B. burgdorferi exhibited OspB polymorphisms with respect to apparent molecular weights and reactivities with monoclonal antibodies. These data suggest that OspB is not a good candidate for vaccination or diagnostic purposes. The present study describes a monoclonal antibody, designated 84C, directed against a very highly conserved domain of the OspB lipoprotein. Western immunoblot analysis with 84C demonstrated reactivity in 84.2% of human, tick, and other vertebrate isolate strains examined from widely diverse geographic regions, including strains of B. burgdorferi sensu stricto and two closely related species, B. garinii and B. afzelii. The 84C-binding region was delimited to a highly conserved 11-amino-acid region in the carboxyl terminus of OspB as demonstrated by (i) DNA sequence analysis of wild-type and 84C-resistant mutant ospB alleles and (ii) deletion mutagenesis of a recombinant ospB gene in Escherichia coli. Finally, the 84C epitope was demonstrated to be exposed on the borrelial surface in situ as (i) the monoclonal antibody 84C was able to agglutinate borrelias in culture and (ii) 84C-resistant escape variants were isolated. These data suggest that the potential value of OspB as a vaccine candidate or diagnostic tool be examined more closely, in the context of the 84C-reactive domain.

Publication Types:
PMID: 7512097 [PubMed - indexed for MEDLINE]

PMCID: PMC263060


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Analysis of Borrelia burgdorferi membrane architecture by freeze-fracture electron microscopy.

Radolf JD, Bourell KW, Akins DR, Brusca JS, Norgard MV.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.

Freeze-fracture electron microscopy was used to investigate the membrane architectures of high-passage Borrelia burgdorferi B31 and low- and high-passage isolates of B. burgdorferi N40. In all three organisms, fractures occurred almost exclusively through the outer membrane (OM), and the large majority of intramembranous particles were distributed randomly throughout the concave OM leaflet. The density of intramembranous particles in the concave OM leaflet of the high-passage N40 isolate was significantly greater than that in the corresponding leaflet of the low-passage N40 isolate. Also noted in the OMs of all three organisms were unusual structures, designated linear bodies, which typically were more or less perpendicular to the axis of the bacterium. A comparison of freeze-fractured B. burgdorferi and Treponema pallidum, the syphilis spirochete, revealed that the OM architectures of these two pathogens differed markedly. All large membrane blebs appeared to be bounded by a membrane identical to the OM of B. burgdorferi whole cells; in some blebs, the fracture plane also revealed a second bilayer closely resembling the B. burgdorferi cytoplasmic membrane. Aggregation of the lipoprotein immunogens outer surface protein A (OspA) and OspB on the bacterial surface by incubation of B. burgdorferi B31 with specific polyclonal antisera did not affect the distribution of OM particles, supporting the contention that lipoproteins do not form particles in freeze-fractured OMs. The expression of poorly immunogenic, surface-exposed proteins as virulence determinants may be part of the parasitic strategy used by B. burgdorferi to establish and maintain chronic infection in Lyme disease.

Publication Types:
PMID: 8282698 [PubMed - indexed for MEDLINE]

PMCID: PMC205010


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Seroprotective groups among isolates of Borrelia burgdorferi.

Lovrich SD, Callister SM, Lim LC, Schell RF.

Wisconsin State Laboratory of Hygiene, Madison.

We demonstrated that different seroprotective groups exist among isolates of Borrelia burgdorferi and Borrelia garinii. The major group was composed of isolates 297, B31, S-1-10, MMTI, IPT, and ATCC 35211 and 21 isolates obtained from California, Illinois, New York, Texas, and Wisconsin. A second group was composed of European isolates PBi and G25. A third group was composed of a single isolate, C-1-11. These groupings were supported by Western immunoblot findings. In addition, the seroprotective groups were confirmed by passive transfer of immune sera and challenge of recipient hamsters with the homologous isolate or other isolates of B. burgdorferi or B. garinii. These studies demonstrate that a monovalent vaccine will not provide complete protection against infection with all isolates of B. burgdorferi.

Publication Types:
PMID: 8406827 [PubMed - indexed for MEDLINE]

PMCID: PMC281168


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Specific adherence of Borrelia burgdorferi extracellular vesicles to human endothelial cells in culture.

Shoberg RJ, Thomas DD.

Department of Periodontics, University of Texas Health Science Center, San Antonio 78284.

Borrelia burgdorferi produces extracellular vesicles which contain some of the outer surface proteins of the bacterium (e.g., OspA and OspB). Borrelial vesicles, isolated by differential centrifugation and filtration, were tested for the ability to bind to cultured human umbilical vein endothelial (HUVE) cells in culture. The recently described lipoprotein OspD was expressed on vesicles. Vesicles exhibited differential expression of OspB and OspD in a relationship with passage number and medium serum supplement type, respectively. Qualitative immunoblotting analyses demonstrated dose-dependent, passage number-dependent adsorption of vesicles by HUVE cells. This adsorption was demonstrated to be dependent upon a borrelial component of the vesicle and not due to the presence of minor contamination with intact spirochetes. Quantitative experiments examining inhibition of B. burgdorferi-HUVE association as a function of prior vesicle-HUVE association demonstrated dependence upon (i) a borrelial component(s) in the vesicle, (ii) low passage number, and (iii) vesicle protein concentration. However, vesicle pretreatment of the HUVE cell monolayer was not requisite for this inhibition. Vesicles from highly passaged borrelias were noninhibitory for B. burgdorferi-HUVE cell association, regardless of the serum used to supplement the medium. The use of vesicles as a tool for studying B. burgdorferi pathogenesis and/or physiology is proposed.

Publication Types:
PMID: 8359911 [PubMed - indexed for MEDLINE]

PMCID: PMC281091


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An OspB mutant of Borrelia burgdorferi has reduced invasiveness in vitro and reduced infectivity in vivo.

Sadziene A, Barbour AG, Rosa PA, Thomas DD.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

Most Borrelia burgdorferi strains have two major surface proteins, OspA and OspB. In the present study, we selected from a clonal population of infectious B. burgdorferi an OspB escape mutant, identified the genetic basis for this phenotype, and evaluated its functional activities. Selection with the anti-OspB antibody H614 was performed in vitro in medium and extended in vivo in scid mice. Mutants with a truncated OspB protein were selected at a frequency of 1 x 10(-5) to 3 x 10(-5). After no major rearrangements in DNA were detected, sequence analysis of the mutant's ospAB locus revealed a single base change in the consensus ribosomal binding sequence for ospB and a single nucleotide deletion in the ospB gene itself. The effect of these mutations was reduced expression of a truncated OspB protein. When functional abilities of the wild type and mutant were compared, the mutant had a threefold-lower capacity to penetrate a human endothelium umbilical vein cell monolayer. Infectivity of wild-type and mutant cells for scid mice was evaluated by culturing different organs, and the median infectious dose was calculated. The inoculum of mutant cells for infecting the mice was 30- to 300-fold higher than that of wild-type cells. This study shows that reduced size and expression of OspB are associated with lowered virulence of B. burgdorferi. Selection of mutants that to some degree remain infectious is one approach to defining the role of different surface proteins in the pathogenesis of Lyme disease.

Publication Types:
PMID: 8359881 [PubMed - indexed for MEDLINE]

PMCID: PMC281052


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Comparative in vitro activities of clarithromycin, azithromycin, and erythromycin against Borrelia burgdorferi.

Dever LL, Jorgensen JH, Barbour AG.

Department of Pathology, University of Texas Health Science Center, San Antonio 78284-7881.

The in vitro activities of the macrolide antibiotics clarithromycin, 14-hydroxy-clarithromycin, azithromycin, and erythromycin against 19 isolates of Borrelia burgdorferi were investigated. MICs ranged from 0.003 to 0.03 microgram of clarithromycin per ml, 0.007 to 0.03 microgram of 14-hydroxyclarithromycin per ml, 0.003 to 0.03 microgram of azithromycin per ml, and 0.007 to 0.06 microgram of erythromycin per ml. Time-kill studies using the B31 strain of B. burgdorferi demonstrated a > or = 3-log10-unit killing after 72 h with each of the macrolide antibiotics tested in concentrations representing twice the respective MICs.

Publication Types:
PMID: 8215288 [PubMed - indexed for MEDLINE]

PMCID: PMC188047


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The biological and social phenomenon of Lyme disease.

Barbour AG, Fish D.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

Lyme disease, unknown in the United States two decades ago, is now the most common arthropod-borne disease in the country and has caused considerable morbidity in several suburban and rural areas. The emergence of this disease is in part the consequence of the reforestation of the northeastern United States and the rise in deer populations. Unfortunately, an accurate estimation of its importance to human and animal health has not been made because of difficulties in diagnosis and inadequate surveillance activities. Strategies for prevention of Lyme disease include vector control and vaccines.

Publication Types:
PMID: 8503006 [PubMed - indexed for MEDLINE]

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In vitro activity of vancomycin against the spirochete Borrelia burgdorferi.

Dever LL, Jorgensen JH, Barbour AG.

Department of Medicine, University of Texas Health Science Center, San Antonio 78284-7881.

Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease, has been reported to be susceptible to a variety of antimicrobial agents. In this investigation, the action of vancomycin, a glycopeptide antibiotic not previously known to have activity against spirochetes, against borrelias was examined. The in vitro activity of vancomycin against a variety of strains of B. burgdorferi and one strain of Borrelia hermsii was determined by use of a microdilution MIC method (L.L. Dever, J.H. Jorgensen, and A.G. Barbour, J. Clin. Microbiol. 30:2692-2697, 1992). MICs ranged from 0.5 to 2 micrograms/ml. MICs of the glycopeptides ristocetin and teicoplanin and the lipopeptide daptomycin against strain B31 of B. burgdorferi were all > or = 8 micrograms/ml. Subsurface plating, time-kill studies, synergy studies, and electron microscopy were used to investigate further the activity of vancomycin against B31. The MBC of vancomycin was 2 micrograms/ml. Time-kill curves demonstrated > or = 3-log10-unit (99.9%) killing of the final inoculum after 72 h by vancomycin concentrations twice the MIC. Synergy between vancomycin and penicillin was demonstrated at concentrations one-fourth the MIC of each drug. In electron microscopy, B31 cells exposed to vancomycin showed a disruption of cellular integrity and were indistinguishable from those exposed to penicillin. These studies demonstrate another class of microorganisms susceptible in vitro to vancomycin.

Publication Types:
PMID: 8517700 [PubMed - indexed for MEDLINE]

PMCID: PMC187913


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The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid.

Sadziene A, Wilske B, Ferdows MS, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.

Borrelia burgdorferi B31 cells lacking all linear plasmids or all but the 49-kb linear plasmid expressed the otherwise silent gene for the outer membrane protein OspC. In the first demonstration of a function for a circular plasmid of Borrelia spp., ospC was located on a 27-kb circular plasmid of B31.

Publication Types:
PMID: 8478109 [PubMed - indexed for MEDLINE]

PMCID: PMC280820


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In vitro inhibition of Borrelia burgdorferi growth by antibodies.

Sadziene A, Thompson PA, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

Function-oriented immunoassays, such as complement fixation and neutralization, are not commonly used in the study of the Lyme disease agent, Borrelia burgdorferi. To determine whether such assays provide information additional to matrix-based methods, such as ELISA, polyclonal antisera and monoclonal antibodies were examined for their abilities to agglutinate viable borreliae and inhibit their in vitro growth in microtiter plates. Different strains of B. burgdorferi in both high and low passage were examined, and the related spirochete Borrelia hermsii and antibodies to it served as controls. Agglutination and complement-independent inhibition of growth with polyclonal sera from rats, mice, and humans and with monoclonal antibodies to outer membrane proteins OspA and OspB was demonstrated. Growth inhibition was obtained with Fab fragments as well as with whole IgG molecules. In comparison with an ELISA using whole cells, the growth inhibition and agglutination assays were generally more specific.

Publication Types:
PMID: 8418163 [PubMed - indexed for MEDLINE]

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Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.

Norris SJ, Carter CJ, Howell JK, Barbour AG.

Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston 77225.

Borrelia burgdorferi, the causative agent of Lyme disease, loses its ability to infect and cause disease in mammalian hosts after repeated in vitro passage. To identify proteins preferentially expressed by the low-passage strain and thus representing potential virulence factors, the polypeptide profiles of virulent, low-passage and nonvirulent, high-passage forms of B. burgdorferi B31 were compared by nonequilibrium pH gradient two-dimensional gel electrophoresis. Four low-passage-associated proteins with relative molecular masses (M(r)s) of 35,000, 28,000, 24,000, and 20,000 were identified. Of these, the 28- and 35-kDa polypeptides were not expressed in detectable quantities in the high-passage B31 strain, whereas the 24- and 20-kDa proteins were present in reduced quantities. All four of these proteins were lipoproteins, as determined by labelling with [3H]palmitate. The abundant 28-kDa component, called outer surface protein D (OspD), is surface exposed on the basis of its proteolysis during treatment of intact organisms with proteinase K. The ospD gene is located on a 38-kb linear plasmid present in seven of nine low-passage strains of B. burgdorferi examined but absent in most high-passage, nonvirulent strains tested. Molecular cloning and sequence analysis of the ospD gene locus revealed an open reading frame encoding a 28,436-Da polypeptide with a putative signal peptidase II leader sequence. An unusual feature of the region upstream of the gene was the presence of seven contiguous, direct repeats of a 17-bp sequence that includes consensus -35 and -10 transcription initiation signals; however, only one transcription initiation site was active as determined by primer extension analysis. Further study of these and other polypeptides associated with low-passage strains may lead to identification of B. burgdorferi gene products required for infection and pathogenesis in mammalian hosts.

Publication Types:
PMID: 1398980 [PubMed - indexed for MEDLINE]

PMCID: PMC258216


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Seroprevalence of antibodies to Borrelia burgdorferi in a population of horses in central Texas.

Cohen ND, Heck FC, Heim B, Flad DM, Bosler EM, Cohen D.

Department of Large Animal Medicine, College of Veterinary Medicine, Texas A&M University, College Station 77843.

Four hundred sixty-nine serum samples were obtained from horses admitted to the internal medicine service of the Texas Veterinary Medical Center between Jan 1 and Dec 31, 1990. Serum samples were tested by ELISA for antibody to Borrelia burgdorferi. Of these 469 samples, 1 (0.2%) was repeatedly seropositive for the organism by ELISA. Confirmatory testing by protein immunoblot was negative. The observed seroprevalence was 0%; the upper bound of the 95% confidence interval was 0.6%. These findings indicate the evidence of infection with B burgdorferi is presently uncommon in horses in central Texas.

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PMID: 1429127 [PubMed - indexed for MEDLINE]

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In vitro antimicrobial susceptibility testing of Borrelia burgdorferi: a microdilution MIC method and time-kill studies.

Dever LL, Jorgensen JH, Barbour AG.

Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284.

The susceptibility of Borrelia burgdorferi, the causative agent of Lyme borreliosis, to various antimicrobial agents varies widely among published studies. These differences are probably due in part to variations in susceptibility testing techniques and growth endpoint determinations. We developed a microdilution method for determining the MICs of antibiotics against B. burgdorferi. The method incorporated BSK II medium, a final inoculum of 10(6) cells per ml, and a 72-h incubation period and was found to be simple and highly reproducible. A variety of antibiotics and strains of B. burgdorferi and one strain of Borrelia hermsii were examined by this method. MICs of penicillin, ceftriaxone, and erythromycin for the B31 strain of B. burgdorferi were 0.06, 0.03, and 0.03 microgram/ml, respectively. We compared the MICs obtained by the microdilution method with those obtained by a macrodilution method using similar criteria for endpoint determinations and found the values obtained by both methods to be in close agreement. To further investigate the bactericidal activities of penicillin, ceftriaxone, and erythromycin against strain B31, we used subsurface plating to determine MBCs and we also performed time-kill studies. The MBCs of penicillin, ceftriaxone, and erythromycin were 0.125, 0.03, and 0.06 micrograms/ml, respectively. Time-kill curves demonstrated a greater than or equal to 3-log10-unit killing after 72 h with penicillin, ceftriaxone, and erythromycin; ceftriaxone provided the greatest reduction in CFU. The described methods offer a more standardized and objective approach to susceptibility testing of B. burgdorferi.

Publication Types:
PMID: 1400969 [PubMed - indexed for MEDLINE]

PMCID: PMC270500


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Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and characterization.

S?dziene A, Rosa PA, Thompson PA, Hogan DM, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

We used polyclonal antisera and monoclonal antibodies (mAbs) to inhibit the growth of clonal populations of two strains of Borrelia burgdorferi, the Lyme disease agent, and thereby select for antibody-resistant mutants. mAbs were directed at the outer membrane proteins, OspA or OspB. Mutants resistant to the growth-inhibiting properties of the antibodies were present in the populations at frequencies ranging from 10(-5) to 10(-2). The several escape variants that were examined were of four classes. Class I mutants were resistant to all mAbs; they lacked OspA and OspB and the linear plasmid that encodes them. Two other proteins were expressed in larger amounts in class I mutants; mAbs to these proteins inhibited the mutant but not the wild-type cells. Class II mutants were resistant to some but not all mAbs; they had truncated OspA and/or OspB proteins. Class III mutants were resistant only to the selecting mAb; they had full-length Osp proteins that were not bound by the selecting antibody in Western blots. In two class III mutants resistant to different anti-OspA mAbs, missense mutations were demonstrated in the ospA genes. Class IV mutants were likewise resistant only to selecting antibody, but in this case the selecting antibody still bound in Western blots.

Publication Types:
PMID: 1339462 [PubMed - indexed for MEDLINE]

PMCID: PMC2119346


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Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Hinnebusch J, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.

Publication Types:
PMID: 1644750 [PubMed - indexed for MEDLINE]

PMCID: PMC206359


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Biological and social determinants of the Lyme disease problem.

Barbour AG.

Department of Medicine, University of Texas Health Science Center, San Antonio 78284.

Lyme disease, presently the most common arthropod-borne disease in the United States, is a phenomenon of both medical and social importance. Notwithstanding the substantial public health threat posed by Borrelia burgdorferi infection, there is reason to suspect that the diagnosis of Lyme disease is made more frequently than justified. On the other hand, the current dissatisfaction with serologic assays for B. burgdorferi infection may lead some physicians to inappropriately abandon consideration of the diagnosis entirely. Either of these outcomes of the patient-physician encounter is to be regretted. The Lyme disease problem, as I call the current situation, has both biological and social determinants.

Publication Types:
PMID: 1365529 [PubMed - indexed for MEDLINE]

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Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi.

Brusca JS, McDowall AW, Norgard MV, Radolf JD.

Department of Microbiology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.

Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracellularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins.

Publication Types:
PMID: 1744059 [PubMed - indexed for MEDLINE]

PMCID: PMC212599


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Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus.

Hinnebusch J, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.

Spirochetes of the genus Borrelia have double-stranded linear plasmids with covalently closed ends. The physical nature of the terminal connections was determined for the 16-kb linear plasmid of the B31 strain of the Lyme disease agent Borrelia burgdorferi. Native telomeric fragments representing the left and right ends of this plasmid were isolated and subjected to Maxam-Gilbert sequence analysis. At the plasmid ends the two DNA strands formed an uninterrupted, perfectly palindromic, AT-rich sequence. This Borrelia linear plasmid consisted of a continuous polynucleotide chain that is fully base paired except for short single-stranded hairpin loops at each end. The left and right telomeres of the 16-kb plasmid were identical for 16 of the first 19 nucleotide positions and constituted an inverted terminal repeat with respect to each other. The left telomere of the 49-kb plasmid of strain B31 was identical to the corresponding telomere of the 16-kb plasmid. Different-sized plasmids of other strains of B. burgdorferi also contained sequences homologous to the left end of the 16-kb plasmid. When the borrelia telomeres were compared with telomeric sequences of other linear double-stranded DNA replicons, sequence similarities were noted with poxviruses and particularly with the iridovirus agent of African swine fever. The latter virus and a Borrelia sp. share the same tick vector. These findings suggest that the novel linear plasmids of Borrelia originated through a horizontal genetic transfer across kingdoms.

Publication Types:
PMID: 1938918 [PubMed - indexed for MEDLINE]

PMCID: PMC209230


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Lipoproteins of Borrelia burgdorferi and Treponema pallidum activate cachectin/tumor necrosis factor synthesis. Analysis using a CAT reporter construct.

Radolf JD, Norgard MV, Brandt ME, Isaacs RD, Thompson PA, Beutler B.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.

Lipoproteins from two pathogenic spirochetes (Borrelia burgdorferi and Treponema pallidum) induced the biosynthesis of TNF in murine macrophages and in permanently transformed macrophages of the cell line RAW 264.7. Induction was studied by measuring the secretion of biologically active TNF and by measuring the activity of the reporter enzyme chloramphenicol acetyltransferase (CAT) produced within macrophages transfected with an endotoxin-responsive CAT construct. Several lines of evidence indicated that the induction of TNF and CAT was attributable to the spirochete lipoproteins rather than to contaminating or endogenous LPS: 1) the dose response curves observed for the lipoproteins were markedly different from those obtained with LPS; 2) lipoprotein-mediated activation was unaffected by amounts of polymyxin B that completely neutralized the induction of TNF and CAT by LPS, 3) low concentrations of the lipoproteins induced TNF in macrophages from endotoxin-unresponsive C3H/HeJ mice as effectively as in macrophages from normal C3H/HeN mice, and 4) isolated spirochete lipoproteins, but not a non-lipoprotein immunogen, were potent inducers of CAT in the transformed macrophages. Moreover, LPS was not detected in the B. burgdorferi lipoprotein mixtures by Limulus amebocyte lysate assay. Proteolytic digestion of the intact bacterial protein preparations only modestly diminished their ability to activate the cells, suggesting that small lipopeptides comprise the biologically active portions of the molecules, as is the case with the murein lipoprotein of Escherichia coli. Through their ability to induce TNF production by macrophages, spirochete lipoproteins may play important roles in the development of the local inflammatory changes and the systemic manifestations that characterize syphilis and Lyme disease.

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PMID: 1890308 [PubMed - indexed for MEDLINE]

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A flagella-less mutant of Borrelia burgdorferi. Structural, molecular, and in vitro functional characterization.

Sadziene A, Thomas DD, Bundoc VG, Holt SC, Barbour AG.

Department of Medicine, University of Texas Health Science Center, San Antonio 78284.

A nonmotile mutant of Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated and characterized. The mutant was compared with the wild-type predecessor as well as with a motile back-revertant of the same genetic background. The mutant lacked, by morphologic, biochemical, and immunologic criteria, the major structural protein of flagella, flagellin. This mutation was not associated with major DNA rearrangements or with failure of transcription. An apparent consequence of a loss of flagella was reduced ability to penetrate human endothelial cell layers in vitro. In another assessment of functional significance, the flagella-less mutant was equal if not superior to flagella-bearing, isogenic isolates when examined in an enzyme-linked immunosorbent assay for anti-B. burgdorferi antibodies in the sera of Lyme disease patients. These studies of a mutant, the first among pathogenic Borrelia spp. to be characterized, indicate that the flagellum and motility it confers play a role in B. burgdorferi's invasion of human tissues. A flagella-less B. burgdorferi may be useful as the basis of a more specific immunoassay and a vaccine for protection against Lyme disease.

Publication Types:
PMID: 2056133 [PubMed - indexed for MEDLINE]

PMCID: PMC296006


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Isolation of Borrelia burgdorferi from arthropods collected in Texas.

Teltow GJ, Fournier PV, Rawlings JA.

Microbiological Services Division, Bureau of Laboratories, Texas Department of Health, Austin.

The Texas Department of Health Laboratory cultured arthropods from November 1988 through December 1989 in an attempt to isolate Borrelia burgdorferi, the etiologic agent of Lyme disease. Spirochetes were isolated from eight of 1,093 pools of arthropods cultured. The spirochetal isolates were from several tick and one flea species, including Amblyomma americanum, A. maculatum, Ixodes scapularis, and Ctenocephalides felis. These 8 isolates reacted specifically when treated with monoclonal antibodies to B. burgdorferi. Polyacrylamide gel electrophoresis of six lysates showed them to be virtually identical with strain B31 of B. burgdorferi.

PMID: 2063950 [PubMed - indexed for MEDLINE]

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Molecular biology of antigenic variation in Lyme borreliosis and relapsing fever: a comparative analysis.

Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

Lyme borreliosis and relapsing fever are human diseases caused by different members of the genus Borrelia. Antigenic variation has been a well-known feature of the pathogenesis of relapsing fever for decades. More recently it has been recognized that Borrelia burgdorferi, the agent of Lyme borreliosis, also can vary its surface antigens. In this review the biology and molecular biology of antigenic variation of the pathogens in these two disorders are compared.

Publication Types:
PMID: 1947817 [PubMed - indexed for MEDLINE]

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Clinical and epizootiologic characteristics of dogs seropositive for Borrelia burgdorferi in Texas: 110 cases (1988).

Cohen ND, Carter CN, Thomas MA Jr, Angulo AB, Eugster AK.

Department of Large Animal Medicine, College of Veterinary Medicine, Texas A & M University, College Station.

Of 2,409 canine serum samples submitted to the Texas Veterinary Medical Diagnostic Laboratory between Jan 1, 1988 and Dec 31, 1988 and tested by immunofluorescent antibody technique for antibody to Borrelia borgdorferi, 132 (5.5%) had positive results. Clinical and epizootiologic characteristics of seropositive dogs from Texas (n = 110) were examined. Male dogs were more likely than female dogs to be seropositive for B burgdorferi. The most frequent clinical sign of disease described in seropositive dogs was lameness; neurologic, ophthalmologic, dermatologic, renal, and hepatic signs also were reported by referring veterinarians.

PMID: 2228777 [PubMed - indexed for MEDLINE]

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Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi.

Hinnebusch J, Bergström S, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.

Publication Types:
PMID: 2388560 [PubMed - indexed for MEDLINE]

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Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

Brandt ME, Riley BS, Radolf JD, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.

The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi.

Publication Types:
PMID: 2318538 [PubMed - indexed for MEDLINE]

PMCID: PMC258571


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Serologic survey of selected zoonotic disease agents in black-tailed jack rabbits from western Texas.

Henke SE, Pence DB, Demarais S, Johnson JR.

Wildlife Management, Texas Tech University, Lubbock 79409.

A serologic survey for the agents of Rocky Mountain spotted fever (RMSF) (Rickettsia rickettsii), Borrelia spp. including the causative agent for Lyme disease (Borrelia burgdorferi), and plague (Yersinia pestis) was conducted on blood samples collected from 30 and 46 black-tailed jack rabbits (Lepus californicus) from an urban environment in Lubbock, Texas (USA) during winter 1987 and the following spring 1988, respectively. Antibody titers to the agents of RMSF and borreliosis were detected in sera of 28 and 1% of the jack rabbits, respectively. Neither organisms (rickettsiae and/or spirochetes) nor their associated antigens were detected in any of the tissue or whole blood samples; plague antibodies were not detected in the 76 jack rabbits sampled. Four of 18 ticks (Dermacentor parumapertus) removed from 12 jack rabbits were positive for RMSF using the fluorescent antibody test. The black-tailed jack rabbit is a common wildlife species living in close proximity to higher density human populations in many areas of the southwestern United States. Our results indicate the potential importance of urban populations of this mammal as reservoirs for at least one important zoonotic disease, RMSF, in western Texas.

PMID: 2106044 [PubMed - indexed for MEDLINE]

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The molecular biology of Borrelia.

Barbour AG.

Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284.

Borrelia burgdorferi, the cause of Lyme disease, has two major outer-membrane proteins, OspA and OspB, which act as surface antigens. A 49-kilobase linear plasmid contains the genes that encode for these surface proteins. Direct examination of denatured plasmid molecules has revealed single-stranded circles with a circumference of approximately 100 kilobases (about twice the length of the linear duplex molecule), a finding that indicates the plasmid strands have covalently closed ends. This form of DNA, while present in eukaryotic organisms and their viruses, has not been observed in a prokaryotic organism. Plasmid heterogeneity has been observed in strains with surface proteins of similar molecular weights and similar monoclonal antibody reactivity. Thus, plasmid analysis may prove a sensitive tool for differentiating strains of B. burgdorferi. Furthermore, since loss of plasmids in vitro has been correlated with loss of the ability of many-passaged strains to cause infection, borrelial plasmids may encode for virulence factors as well.

Publication Types:
PMID: 2682959 [PubMed - indexed for MEDLINE]

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Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorferi.

Bundoc VG, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

The outer membrane protein OspB of Borrelia burgdorferi, the Lyme borreliosis agent, differs in relative molecular weight (Mr) among strains. To determine whether antigenic variation occurs in B. burgdorferi, a cell population of the human isolate HB19 was cloned first by being diluted in broth and then by being plated on agar medium. Several clones were obtained and characterized by polyacrylamide gel electrophoresis, in situ protease treatment, and Western (immunoblot), Southern, and Northern (RNA) blot analyses. Variants featuring OspB proteins that differed in Mrs and in reactivities with monoclonal antibodies were found. One variant made increased amounts of a 21,000-molecular-weight protein (21K protein) in addition to normal amounts of a 33K OspB protein. Another variant did not produce an OspB protein at all but did express an 18.5K protein. Both the 18.5K and 21K proteins were susceptible in situ to trypsin and were bound by a monoclonal antibody directed against the OspB of strain HB19. There were no differences in the Southern and Northern blot analyses of the different variants. The results led to the following conclusions. (i) Clonal polymorphisms in the surface protein OspB occurred in B. burgdorferi. (ii) Hitherto uncharacterized 18.5K and 21K proteins were protease susceptible, antigenically related to OspB, and apparently produced in greater amounts when an OspB either was not produced or was altered in structure. (iii) The OspB variations, including its absence from cells, were not accounted for by major DNA rearrangements or failure of transcription of the ospB gene.

Publication Types:
PMID: 2668185 [PubMed - indexed for MEDLINE]

PMCID: PMC313519


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Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent.

Ferdows MS, Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

Using pulsed-field gel electrophoresis we examined the genome of Borrelia burgdorferi, a eubacterium of the spirochete phylum and the agent of Lyme disease. A population of this species' cells was lysed in situ in agarose blocks. An abundant DNA form that behaved as a linear duplex molecule under different electrophoretic conditions was found. The estimated size of the molecule was 950 kilobases. DNA from two other genera of spirochetes did not enter the gel under these conditions. These studies indicate that Borrelia spirochetes, perhaps uniquely among prokaryotic organisms, have linear chromosomes.

Publication Types:
PMID: 2762306 [PubMed - indexed for MEDLINE]

PMCID: PMC297753


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Borrelia sp. infection in coyotes, black-tailed jack rabbits and desert cottontails in southern Texas.

Burgess EC, Windberg LA.

Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison 53706.

Coyotes (Canis latrans) from southern Texas were sampled for antibodies to Borrelia burgdorferi from 1980 to 1986; black-tailed jack rabbits (Lepus californicus) and desert cottontails (Sylvilagus audubonii) were sampled in 1986. Coyote fetuses, adult coyote kidneys, and black-tailed jack rabbit and desert cottontail kidneys were cultured for B. burgdorferi in 1986. Results of indirect immunofluorescent antibody (IFA) tests for B. burgdorferi in coyotes were as follows (number positive at a dilution of greater than or equal to 1:128/number tested): 1980 (0 of 30), 1981 (0 of 21), 1982 (0 of 53), 1983 (0 of 78), 1984 (47 of 97), 1985 (20 of 88), and 1986 (42 of 80). Eight of 26 black-tailed jack rabbits and two of seven desert cottontails tested in 1986 had IFA titers to B. burgdorferi of greater than or equal to 1:128. Borrelia burgdorferi was isolated from one of five coyote fetuses, three of 31 adult coyote kidneys, and two of 10 black-tailed jack rabbit kidneys in 1986. These results indicate that B. burgdorferi infection has been present in coyotes in Texas, at least since 1984 and that transplacental transmission occurs.

Publication Types:
PMID: 2644452 [PubMed - indexed for MEDLINE]

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Broad-host-range plasmid and M13 bacteriophage-derived vectors for promoter analysis in Escherichia coli and Pseudomonas aeruginosa.

Konyecsni WM, Deretic V.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

A set of bacteriophage and plasmid vectors containing xylE as a reporter gene was constructed for the analysis of promoters functional in Escherichia coli and in other Gram-negative bacteria. Two M13 bacteriophage derivatives, M13mVDX18 and M13mMK010, were designed for rapid cloning, screening and sequencing of DNA fragments promoting transcription in E. coli. To demonstrate their utility, total cellular DNA from a variety of bacterial species including Pseudomonas aeruginosa strain PAO was shotgun cloned in M13 vectors and clones displaying promoter activity in E. coli were isolated. These randomly cloned promoters from P. aeruginosa, Borrelia burgdorferi, Streptococcus pneumoniae and other bacterial species were sequenced without a need for further subcloning manipulation. The promoter activity of P. aeruginosa clones was verified by subcloning inserts on a broad-host-range promoter probe vector pVDX18 and assaying the xylE transcription from these promoters in P. aeruginosa. The pVDX18 vector was also used for initial characterization of the algD promoter controlling mucoidy in P. aeruginosa. The activities of the wild-type and deletion clones of the algD promoter were compared. Results indicated that the region containing direct and inverted repeats at -55 to -110 bp upstream of the mRNA 5' end was important for the activation of the algD transcription in mucoid P. aeruginosa infecting cystic fibrosis patients.

Publication Types:
PMID: 3149945 [PubMed - indexed for MEDLINE]

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Laboratory aspects of Lyme borreliosis.

Barbour AG.

Department of Medicine, University of Texas Health Science Center, San Antonio 78284.

Lyme borreliosis (Lyme disease), a common tick-borne disorder of people and domestic animals in North America and Europe, is caused by the spirochete Borrelia burgdorferi. Following the discovery and initial propagation of this agent in 1981 came revelations that other tick-associated infectious disorders are but different forms of Lyme borreliosis. A challenge for the clinician and microbiology laboratory is confirmation that a skin rash, a chronic meningitis, an episode of myocarditis, or an arthritic joint is the consequence of B. burgdorferi infection. The diagnosis of Lyme borreliosis may be established by (i) directly observing the spirochete in host fluid or tissue, (ii) recovering the etiologic spirochete from the patient in culture medium or indirectly through inoculation of laboratory animals, or (iii) carrying out serologic tests with the patient's serum or cerebrospinal fluid. The last method, while lacking in discriminatory power, is the most efficacious diagnostic assay for most laboratories at present.

Publication Types:
PMID: 3069200 [PubMed - indexed for MEDLINE]

PMCID: PMC358062


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Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent.

Barbour AG.

Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.

A simple procedure for extraction of plasmid-enriched DNA from borreliae was used in a plasmid analysis of 13 strains of the Lyme disease agent, Borrelia burgdorferi. The extracted DNA was subjected to low-percentage agarose gel electrophoresis and examined either directly by ethidium bromide staining or after hybridization of the plasmids in situ with a DNA probe for the gene encoding the major outer membrane protein OspA. Each isolate had four to seven discernible plasmids of various sizes. Only 2 of the 13 strains had the same plasmid profile. The ospA gene probe hybridized to large plasmids to strains from both North America and Europe. A strain which had been passaged many times was found to have lost two of the six plasmids originally present. These findings indicate the potential usefulness of plasmid analysis as a strain-typing procedure and for identifying possible plasmid-conferred virulence factors.

Publication Types:
PMID: 3356787 [PubMed - indexed for MEDLINE]

PMCID: PMC266316


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The genes encoding major surface proteins of Borrelia burgdorferi are located on a plasmid.

Barbour AG, Garon CF.

Department of Medicine, University of Texas Health Science Center, San Antonio 78284.

Publication Types:
PMID: 3190089 [PubMed - indexed for MEDLINE]

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Isolation of Borrelia spirochetes from patients in Texas.

Rawlings JA, Fournier PV, Teltow GJ.

The Texas Department of Health Laboratory began culturing the Lyme disease spirochete Borrelia burgdorferi in 1985. This organism was subsequently isolated from blood, cerebrospinal fluid, joint fluid, skin, bone, and autopsy tissues from humans. Fluorescent-antibody tests with murine monoclonal antibodies confirmed that seven of these isolates were B. burgdorferi and that two others belonged to the genus Borrelia.

PMID: 3611307 [PubMed - indexed for MEDLINE]

PMCID: PMC269164



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THE DECLINE OF A GIFTED AND SINCERE MAN

LIFETIME LYME EXPOSURES AND OTHER
TICK-BORNE INFECTIONS AND PRESIDENT BUSH

I recently was talking to a passionate Republican who was discussing the decreased ability of President Bush and his decreased memory, cognitive processing speed and speech fluency.

He had great respect for the President. He also felt very strongly something was going on that was missed.

Simply, the President has had behavior and cognition and emotional issues on and off for his entire life. He has had massive deer tick exposures. His huge EM bulls-eye rash was handled curiously and was assumed to be the only infection.

*****

Does Lyme Disease Explain Bush's Erratic Behavior?

Posted by Larkin
Published: Aug 10, 07 06:00 AM

The White House has revealed that President Bush received undisclosed treatment for Lyme Disease in August of 2006 as reported by ABC. Lyme Disease (Borreliosis) is a bacterial infection that is typically acquired by the bite of an infected tick. Wikipedia has the gory details of this ugly disease: The disease varies widely in its presentation, which may include a rash and flu-like symptoms in its initial stage, followed by the possibility of musculoskeletal, arthritic, neurologic, psychiatric and cardiac manifestations. In most cases of Lyme disease, symptoms can be eliminated with antibiotics, especially if treatment is begun early in the course of illness.

A percentage of patients with Lyme disease have symptoms that last months to years after treatment with antibiotics. These symptoms can include muscle and joint pains, arthritis, stiff neck, cognitive defects, neurological complaints or fatigue. The cause of these continuing symptoms is not yet known. There is some evidence that they may result from an autoimmune type of response, in which a person's immune system continues to respond even after the infection has been cleared, as well as evidence of ongoing infection with the spirochete.

The White House says that Bush was treated for "early, localized Lyme disease" after developing the characteristic bullseye rash. They provided a lame excuse for why Bush's disease was not disclosed earlier and then gave us the remarkable news that doctors have decided not to perform blood tests to determine the extent of his illness: White House spokesman Scott Stanzel said Bush's treatment was not disclosed earlier because it happened after his last physical, on Aug. 1, 2006. He said doctors decided not to perform blood tests for Lyme disease because the treatment worked for the one area where the president experienced a rash, and he never progressed to other symptoms or saw a recurrence.

I don't know about you but if I was in Bush's situation I certainly would have had the full battery of blood tests to determine the extent of my affliction with the disease. It certainly is bizarre that Bush's doctors would elect not to conduct such tests when there is clearly no downside to doing so. Why wouldn't they want to be absolutely certain about the extent of the disease? Especially, when the patient is the President of the United States?

The only answer I can come up with is that Bush's doctors already know how bad it is because he has had the disease for a number of years. The President's bizarre behavior during press conferences might be explained by the neurological and cognitive damage of the disease.

It certainly is disquieting that the White House thought it necessary to cover up the fact that Bush has been stricken with this serious disease. And for what reason? Actions like these will only create suspicion that the disease is worse than they are letting on.

wizbangblue.com/2007/08/10/does-lyme-disease-explain-bushs-erratic-behavior.php

Washington Post Quotes

A report of the president's recent medical examination said his case had "complete resolution" and was "without recurrence" since being treated last August. The illness, an infection carried by deer ticks that is prevalent in the Northeastern United States, had not been previously revealed.

While untreated Lyme disease can cause arthritis, an abnormal heart rhythm and problems with the nervous system, those complications usually can be prevented by taking antibiotics at an early stage of the infection. The medical record did not describe the details of the president's therapy.

Up to 15 percent of people treated for Lyme disease later complain of symptoms such as fatigue and muscle pain. Whether that is a consequence of the infection is uncertain and a matter of controversy. Chronic pain and tiredness are extremely common in adults; whether people who have had Lyme disease suffer from those problems in higher numbers is unknown.

"I wouldn't expect any problem at all for the president," said Gary Wormser, chief of infectious diseases at New York Medical College and an expert on Lyme disease. "He won't be impacted by this infection in the future."

Lyme disease, named after the town in Connecticut where the first cases were identified in the 1970s, causes a rash that is often its sole manifestation. Classically it is a large reddish oval with a lighter-colored center and is often described as looking like a target.

White House spokesman Scott Stanzel said Bush found a rash on the front of his lower left leg and alerted White House physicians.

While the Lyme organism Borrelia burgdorferi can sometimes be isolated in the skin or bloodstream — and antibodies to it can also eventually be detected in the blood — laboratory testing is often not done. That is because a person with a typical rash and a history of outdoor activity will be treated for the disease, regardless of what the tests show.

Without such tests, however, it is impossible to rule out a Lyme disease look-alike called STARI as the cause of the president's illness last summer. STARI stands for "Southern tick-associated rash illness." It also causes a target-like rash and is associated with a tick bite, but the causative organism has not been found.

STARI is common in Texas. The lone star tick is the species that transmits it. There are no documented cases of Lyme disease in the president's home state, where he spent much of last August on vacation.

[REALLY? THE VETS SURE REPORT DIFFERENT, AND MANY PATIENTS HAVE POSITIVE LYME TESTS, BABESIA TESTS AND EHRLICHIA TESTS AND BARTONELLA TESTS IN TEXAS.]

http://www.washingtonpost.com/wp-dyn/content/article/2007/08/08/AR2007080802268.html
[For actual knowledge of STARI read Dr. Masterson. Period].

****

Am. J. Trop. Med. Hyg., 44(5), 1991, pp. 469-474

Copyright © 1991 by The American Society of Tropical Medicine and Hygiene

Isolation of Borrelia burgdorferi from arthropods Collected in Texas

Glenna J. Teltow, Paul V. Fournier and Julie A. Rawlings

Microbiological Services Division, Bureau of Laboratories; and Zoonosis Control Division, Bureau of Veterinary Public Health, Texas Department of Health, Austin, Texass

The Texas Department of Health Laboratory cultured arthropods from November 1988 through December 1989 in an attempt to isolate Borrelia burgdorferi, the etiologic agent of Lyme disease. Spirochetes were isolated from eight of 1,093 pools of arthropods cultured. The spirochetal isolates were from several tick and one flea species, including Amblyomma americanum, A. maculatum, Ixodes scapularis, and Ctenocephalides felis. These 8 isolates reacted specifically when treated with monoclonal antibodies to B. burgdorferi. Polyacrylamide gel electrophoresis of six lysates showed them to be virtually identical with strain B31 of B. burgdorferi.

  • In Texas, there are 11 public health regions. Patients with Lyme disease reside in every public health region in Texas.
  • Borrelia burgdorferi, the agent of Lyme disease has been detected in Texas ticks.
  • Epidemiological evidence suggests Amblyomma americanum, the "Lone Star" tick, is the vector of Lyme disease in Texas. This is an aggressive species that will feed on a variety of hosts including humans. In a Texas Department of Health study conducted in 1990 and 1991, A. americanum ticks were gathered from nine Texas areas. Of the over 28,000 ticks collected, 26,901 or 95% were A. americanum. Visitors to any area with high vegetation are at considerable risk of being bitten by lone star ticks and are at risk of acquiring Lyme disease.
  • There are four reportable tick-borne illnesses in Texas: ehrlichiosis, Lyme disease, rocky mountain spotted fever and tick-borne relapsing fever. Patients in Texas have also been diagnosed with babesiosis, a malaria-like, tick-borne illness with recurring fevers.
  • Failure to report is a Class B misdemeanor under the Texas Health and Safety Code, Section 81.049, but this provision is rarely, if ever, enforced.
  • Texas is a passive surveillance state; and it is likely that there is considerable underreporting of tick-borne illnesses.

www.txlda.org/facts.htm

*****

Journal of Wildlife Diseases, 25(1), 1989, pp. 47-51
© Wildlife Disease Association 1989

Borrelia sp. infection in coyotes, black-tailed jack rabbits and desert cottontails in southern Texas

EC Burgess and LA Windberg

ABSTRACT

Coyotes (Canis latrans) from southern Texas were sampled for antibodies to Borrelia burgdorferi from 1980 to 1986; black-tailed jack rabbits (Lepus californicus) and desert cottontails (Sylvilagus audubonii) were sampled in 1986. Coyote fetuses, adult coyote kidneys, and black-tailed jack rabbit and desert cottontail kidneys were cultured for B. burgdorferi in 1986. Results of indirect immunofluorescent antibody (IFA) tests for B. burgdorferi in coyotes were as follows (number positive at a dilution of greater than or equal to 1:128/number tested): 1980 (0 of 30), 1981 (0 of 21), 1982 (0 of 53), 1983 (0 of 78), 1984 (47 of 97), 1985 (20 of 88), and 1986 (42 of 80). Eight of 26 black-tailed jack rabbits and two of seven desert cottontails tested in 1986 had IFA titers to B. burgdorferi of greater than or equal to 1:128. Borrelia burgdorferi was isolated from one of five coyote fetuses, three of 31 adult coyote kidneys, and two of 10 black-tailed jack rabbit kidneys in 1986. These results indicate that B. burgdorferi infection has been present in coyotes in Texas, at least since 1984 and that transplacental transmission occurs.

***

Results: During 1992?1998, a total of 88,967 cases of Lyme disease was reported to CDC by 49 states and the District of Columbia.

www.cdc.gov/mmwr/PDF/ss/ss4903.pdf


Sample Lyme and Other Tick Infection Work
Completed in Texas Institutions
or Reporting Infection in TX

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sodA is essential for virulence of Borrelia burgdorferi in the murine model of Lyme disease.

Esteve-Gassent MD, Elliott NL, Seshu J.

South Texas Center for Emerging Infectious Diseases and Department of Biology, The University of Texas at San Antonio, San Antonio, TX-78249.

Abstract Borrelia burgdorferi, the causative agent of Lyme disease, has a limited set of genes to combat oxidative/nitrosative stress encountered in its tick vector or mammalian hosts. We inactivated the gene encoding for superoxide dismutase A (sodA, bb0153), an enzyme mediating the dismutation of superoxide anions and examined the in vitro and in vivo phenotype of the mutant. There were no significant differences in the in vitro growth characteristics of the sodA mutant compared to the control strains. Microscopic analysis of viability of spirochetes revealed greater percentage of cell death upon treatment of sodA mutant with superoxide generators compared to its controls. Infectivity analysis in C3H/HeN mice following intradermal needle inoculation of 10(3) or 10(5) spirochetes per mouse revealed complete attenuation of infectivity for the sodA mutant compared to control strains at 21 days post-infection. The sodA mutant was more susceptible to the effects of activated macrophages and neutrophils suggesting that its in vivo phenotype is partly due to the killing effects of activated immune cells. These studies indicate that SodA plays an important role in combating oxidative stress and is essential for the colonization and dissemination of B. burgdorferi in the murine model of Lyme disease.

PMID: 19040638 [PubMed - as supplied by publisher]

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Borrelia burgdorferi lacking DbpBA exhibits an early survival defect during experimental infection.

Weening EH, Parveen N, Trzeciakowski JP, Leong JM, Höök M, Skare JT.

Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.

Several Borrelia burgdorferi genes induced under mammalian host conditions have been purported to be important in Lyme disease pathogenesis based on their binding to host structures. These genes include the dbpBA locus, whose products bind host decorin and glycosoaminoglycans. Recently, the dbpBA genes were reported to be involved in borrelial infectivity. Here we extended the previous observations by using culture and quantitative PCR to evaluate low- and high-dose murine infection by a Delta dbpBA::Gent(r) derivative of B. burgdorferi strain B31. The results indicate that the Delta dbpBA::Gent(r) mutant is attenuated in the ability to initially colonize and then persist in multiple tissues. The mutant exhibited a colonization defect as early as 3 days postinfection, before the development of an adaptive immune response, and after low-dose infection of SCID mice, which are deficient in adaptive immunity. These findings suggest that the inability to adhere to host decorin may promote clearance of B. burgdorferi, presumably via innate immune mechanisms. In a high-dose infection, the mutant disseminated to several tissues, particularly joint tissue, but it was generally cleared from these tissues by 3 weeks postinfection. Finally, following high-dose infection of SCID mice, the dbpBA mutant exhibited only a mild colonization defect, suggesting that the adaptive response is involved in the clearance of the mutant in immunocompetent mice. Taken together, these results suggest that the DbpBA proteins facilitate the colonization of multiple tissues by B. burgdorferi and are required for optimal resistance to both innate and adaptive immune mechanisms following needle inoculation.

Publication Types:
PMID: 18809667 [PubMed - in process]

PMCID: PMC2583571 [Available on 2009/06/01]


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Deletion of BBA64, BBA65, and BBA66 loci does not alter the infectivity of Borrelia burgdorferi in the murine model of Lyme disease.

Maruskova M, Seshu J.

South Texas Center for Emerging Infectious Diseases and Department of Biology, The University of Texas at San Antonio, San Antonio, Texas 78249, USA.

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its tick vector versus vertebrate hosts. Whole-genome transcriptional profile analysis of B. burgdorferi, propagated in vitro under mammalian-host-specific conditions, revealed significant upregulation of several linear plasmid 54 (lp54)-encoded open reading frames (ORFs). Among these ORFs, BBA64, BBA65, and BBA66 have been shown to be upregulated in response to multiple mammalian-host-specific signals. Recently, we determined that there was no significant difference in the ability of BBA64(-) mutant to infect C3H/HeN mice compared to its isogenic control strains, suggesting that B. burgdorferi might utilize multiple, functionally related determinants to establish infection. We further generated BBA65(-) and BBA66(-) single mutants in a noninfectious, lp25(-) clonal isolate of B. burgdorferi strain B31 (ML23) and complemented them with the minimal region of lp25 (BBE22) required for restoring the infectivity. In addition, we generated a BBA64(-) BBA65(-) BBA66(-) triple mutant using an infectious, clonal isolate of B. burgdorferi strain B31 (5A11) that has all of the infection-associated plasmids. There were no significant differences in the ability to isolate viable spirochetes from different tissues of C3H/HeN mice infected via intradermal needle inoculation with either the individual single mutants or the triple mutant compared to their respective isogenic parental strains at days 21 and 62 postinfection. These observations suggest that B. burgdorferi can establish infection in the absence of expression of BBA64, BBA65, and BBA66 in the murine model of Lyme disease.

Publication Types:
PMID: 18765733 [PubMed - indexed for MEDLINE]

PMCID: PMC2573326 [Available on 2009/03/01]


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Transcriptional interplay among the regulators Rrp2, RpoN and RpoS in Borrelia burgdorferi.

Ouyang Z, Blevins JS, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

The RpoN-RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN-RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.

Publication Types:
PMID: 18757798 [PubMed - indexed for MEDLINE]

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Assessment of decorin-binding protein A to the infectivity of Borrelia burgdorferi in the murine models of needle and tick infection.

Blevins JS, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. jon.blevins@utsouthwestern.edu

BACKGROUND: Decorin-binding proteins (Dbps) A and B of Borrelia burgdorferi, the agent of Lyme disease, are surface-exposed lipoproteins that presumably bind to the extracellular matrix proteoglycan, decorin. B. burgdorferi infects various tissues including the bladder, heart, joints, skin and the central nervous system, and the ability of B. burgdorferi to bind decorin has been hypothesized to be important for this disseminatory pathogenic strategy. RESULTS: To determine the role of DbpBA in the infectious lifecycle of B. burgdorferi, we created a DbpBA-deficient mutant of B. burgdorferi strain 297 and compared the infectious phenotype of the mutant to the wild-type strain in the experimental murine model of Lyme borreliosis. The mutant strain exhibited a 4-log decrease in infectivity, relative to the wild-type strain, when needle inoculated into mice. Upon complementation of the DbpBA-mutant strain with DbpA, the wild-type level of infectivity was restored. In addition, we demonstrated that the DbpBA-deficient mutant was able to colonize Ixodes scapularis larval ticks after feeding on infected mice and persist within the ticks during the molt to the nymphal state. Moreover, surprisingly, the DbpBA-mutant strain was capable of being transmitted to naïve mice via tick bite, giving rise to infected mice. CONCLUSION: These results suggest that DbpBA is not required for the natural tick-transmission process to mammals, despite inferences from needle-inoculation experiments implying a requirement for DbpBA during mammalian infection. The combined findings also send a cautionary note regarding how results from needle-inoculation experiments with mice should be interpreted.

Publication Types:
PMID: 18507835 [PubMed - indexed for MEDLINE]

PMCID: PMC2430964


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Ultrastructural evidence of the ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with Rickettsia, Borrelia, and a flavivirus.

Popov VL, Korenberg EI, Nefedova VV, Han VC, Wen JW, Kovalevskii YV, Gorelova NB, Walker DH.

Department of Pathology, The University of Texas Medical Branch, Center for Biodefense and Emerging Infectious Diseases, Galveston, Texas 77555, USA.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

Publication Types:
PMID: 18171109 [PubMed - indexed for MEDLINE]

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Role of the BBA64 locus of Borrelia burgdorferi in early stages of infectivity in a murine model of Lyme disease.

Maruskova M, Esteve-Gassent MD, Sexton VL, Seshu J.

South Texas Center for Emerging Infectious Diseases and Department of Biology, The University of Texas at San Antonio, San Antonio, Texas 7824, USA.

Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.

Publication Types:
PMID: 17984202 [PubMed - indexed for MEDLINE]

PMCID: PMC2223643


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Spirochetemia caused by Borrelia turicatae infection in 3 dogs in Texas.

Whitney MS, Schwan TG, Sultemeier KB, McDonald PS, Brillhart MN.

Texas Veterinary Medical Diagnostic Laboratory, Texas A&M University, Amarillo, TX, USA. whitneym@missouri.edu

Spirochetemia was diagnosed in 2 Siberian Huskies and a Rottweiler from the northwestern region of Texas between June 1999 and October 2001. Clinical findings were nonspecific; tick exposure was documented in 2 of the dogs. Hematologic abnormalities included anemia (n=2), neutrophilia (n=2, including 1 with a left shift), lymphopenia (n=3), eosinopenia (n=3), and thrombocytopenia (n=2). One anemic dog had a positive Coombs' test. In 1 dog, Western blot analysis of serum yielded multiple positive bands with B turicatae lysate, indicating the spirochetemia most likely was due to B turicatae infection. In 2 dogs, spirochetes were cultured from the blood and identified using DNA analysis as Borrelia turicatae; 1 of these dogs also was seropositive for Ehrlichia canis and B burgdorferi. In 2 cases, spirochetemia was more prominent in blood smears prepared immediately after sample collection than in smears prepared from EDTA blood. Two dogs recovered with doxycycline treatment; 1 dog declined clinically despite treatment and was euthanized. B turicatae is the agent of tick-borne (endemic) relapsing fever in humans and is distinct from B burgdorferi, the agent of Lyme disease; however, serologic cross-reactivity may occur. B turicatae is transmitted by the soft tick, Ornithodoros turicata, and infection should be considered in dogs with spirochetemia and possible exposure to the tick vector.

Publication Types:
PMID: 17523100 [PubMed - indexed for MEDLINE]

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Split target specificity of ResT: a design for protein delivery, site selectivity and regulation of enzyme activity?

Jayaram M.

Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA. jayaram@icmb.utexas.edu

The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.

Publication Types:
PMID: 17462008 [PubMed - indexed for MEDLINE]

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Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi.

Blevins JS, Revel AT, Smith AH, Bachlani GN, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9048, USA.

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.

Publication Types:
PMID: 17220265 [PubMed - indexed for MEDLINE]

PMCID: PMC1828772


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Evidence that RpoS (sigmaS) in Borrelia burgdorferi is controlled directly by RpoN (sigma54/sigmaN).

Smith AH, Blevins JS, Bachlani GN, Yang XF, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.

The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.

Publication Types:
PMID: 17158681 [PubMed - indexed for MEDLINE]

PMCID: PMC1855718


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Borrelia burgdorferi alters its gene expression and antigenic profile in response to CO2 levels.

Hyde JA, Trzeciakowski JP, Skare JT.

Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.

The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO(2), serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO(2), 3% H(2), 0.087 ppm O(2)) and microaerophilic (1% CO(2), 3.48 ppm O(2)) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO(2). In this study we identified CO(2) as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO(2), independent of O(2) levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO(2). Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO(2), indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO(2) may be an additional cue for borrelial host adaptation and gene regulation.

Publication Types:
PMID: 17098904 [PubMed - indexed for MEDLINE]

PMCID: PMC1797391


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Identification of potential virulence determinants by Himar1 transposition of infectious Borrelia burgdorferi B31.

Botkin DJ, Abbott AN, Stewart PE, Rosa PA, Kawabata H, Watanabe H, Norris SJ.

Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77225-0708, USA.

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

Publication Types:
PMID: 17015459 [PubMed - indexed for MEDLINE]

PMCID: PMC1698074


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Transcriptional profiling of Borrelia burgdorferi containing a unique bosR allele identifies a putative oxidative stress regulon.

Hyde JA, Seshu J, Skare JT.

Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Science Center, College Station, 77843-1114, USA.

Borrelia burgdorferi regulates gene expression in response to environmental conditions, including temperature, pH, redox potential and host factors. B. burgdorferi encodes a PerR homologue designated BosR, which presumably serves as a global regulator of genes involved in the oxidative stress response. Infectious B. burgdorferi strain B31 is resistant to oxidative stressors in vitro, whereas the non-infectious isolate was sensitive due, in part, to a point mutation that converts an arginine to a lysine at residue 39 of BosR. Subsequent insertional inactivation of this bosRR39K allele (bosRR39K : : kan(R)) restored resistance to oxidative stressors. These observations suggest that the B. burgdorferi non-infectious bosRR39K : : kan(R) strain may transcribe genes that are also expressed in infectious B. burgdorferi cells, but are repressed in the bosRR39K background, thus explaining the different oxidative stress phenotypes observed between these isolates. To test this hypothesis, macroarray technology and quantitative RT-PCR were utilized to compare the transcriptional profiles from the isogenic bosRR39K and bosRR39K : : kan(R) isolates. Array data indicated that 88 ORFs were significantly expressed in the absence of BosRR39K. Since most affected genes mapped to the chromosome, it is likely that these genes define an important physiologic response for B. burgdorferi. Included within the genes identified was the detoxification gene sodA, as well as other loci not overtly linked to oxidative stress. These results suggest that a putative BosR regulon, as defined by the bosRR39K allele, is required to combat toxic oxidative intermediates, but may also be involved in adaptive strategies that are independent of reactive oxygen species.

Publication Types:
PMID: 16946255 [PubMed - indexed for MEDLINE]

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Differential effects of alcohols on conformational switchovers in alpha-helical and beta-sheet protein models.

Perham M, Liao J, Wittung-Stafshede P.

Department of Chemistry, Keck Center for Structural Computational Biology, Rice University, 6100 Main Street, Houston, Texas 77251, USA.

Organic solvents may induce non-native structures of proteins that mimic folding intermediates and/or conformations that occur in proximity to biological membranes. Here we systematically investigate the effects of simple (i.e., MeOH and EtOH) and fluorinated (i.e., trifluoroethanol, TFE) alcohols on the secondary structure and thermodynamic stability of two complementary model proteins using a combination of circular dichroism, fluorescence, and Fourier transform infrared (FTIR) detection methods. The selected proteins are alpha-helical Borrelia burgdorferi VlsE and beta-sheet human mitochondrial co-chaperonin protein 10 (cpn10). We find that switches between VlsE's native and non-native superhelical and beta-sheet structures readily occur (pH 7, 20 degrees C). The pathway depends on the alcohol: addition of MeOH induces a transition to a superhelical structure that is followed by conversion to beta-structure, whereas EtOH only unfolds the protein. TFE unfolds VlsE at low percentages but promotes the formation of a superhelical state upon further additions. For cpn10, both MeOH and TFE additions govern initial unfolding; however, further additions of MeOH result in the formation of a non-native beta-structure, whereas subsequent additions of TFE induce a superhelical structure. EtOH additions promptly unfold and precipitate cpn10. Both VlsE's and cpn10's non-native structures exhibit high stability toward chemical and thermal perturbations. This study demonstrates that in response to different alcohols, polypeptides can readily adopt both alpha- and beta-enriched conformations. The biological significance of these findings is discussed.

Publication Types:
PMID: 16784225 [PubMed - indexed for MEDLINE]

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Detection of Babesia and Anaplasma species in rabbits from Texas and Georgia, USA.

Yabsley MJ, Romines J, Nettles VF.

Southeastern Cooperative Wildlife Disease Study and Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA. myabsley@uga.edu

Rabbits have been shown to harbor a suite of zoonotic organisms, including a Babesia species, Borrelia burgdorferi, and Anaplasma phagocytophilum. In this study, we conducted a molecular survey for various tick-borne pathogens in three species of rabbits from Texas and Georgia. Of 18 black-tailed jackrabbits (Lepus californicus) tested from Texas, six (28%) were polymerase chain reaction (PCR) positive for Babesia, and nucleotide sequencing revealed two distinct species or strains. Two jackrabbits were infected with a Babesia species or strain (Babesia sp. A) that was nearly identical (99.9%) to a piroplasm previously detected in humans from Washington state, and the remaining four jackrabbits were infected with a Babesia species (Babesia sp. B) that was most similar (99.7%) to a Babesia species detected in cottontail rabbits from Massachusetts and humans from Kentucky and Missouri. Eleven (61%) black-tailed jackrabbits were positive for A. bovis, and one was positive for A. phagocytophilum. Two of four desert cottontails (Sylvilagus audubonii) from Texas were positive for the Babesia sp. B, and one desert cottontail each was positive for A. bovis and A. phagocytophilum. One of these desert cottontails was coinfected with the Babesia sp. B and A. phagocytophilum, and five jackrabbits were coinfected with Babesia species and A. bovis. Of 19 eastern cottontails (S. floridanus) from Georgia, only one (5.3%) was positive for A. phagocytophilum, and three (15.8%) were positive for A. bovis. No rabbits from Texas or Georgia were positive for Borrelia species. The only tick species detected on the Texas and Georgia rabbits was the rabbit tick, Haemaphysalis leporispalustris. These data extend the geographic and host range of these pathogens, and because both the Babesia species and A. phagocytophilum are potential zoonotic pathogens, it is important to be aware that these organisms are enzootic in parts of the southern United States.

Publication Types:
PMID: 16584322 [PubMed - indexed for MEDLINE]

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The dynamic proteome of Lyme disease Borrelia.

Norris SJ.

Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, TX 77225-0708, USA. Steven.J.Norris@uth.tmc.edu

The proteome of the spirochete bacterium Borrelia burgdorferi, the tick-borne agent of Lyme disease, has been characterized by two different approaches using mass spectrometry, providing a launching point for future studies on the dramatic changes in protein expression that occur during transmission of the bacterium between ticks and mammals.

Publication Types:
PMID: 16563176 [PubMed - indexed for MEDLINE]

PMCID: PMC1557748


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The mystery of Morgellons disease: infection or delusion?

Savely VR, Leitao MM, Stricker RB.

South Austin Family Practice Clinic, Austin, Texas, USA.

Morgellons disease is a mysterious skin disorder that was first described more than 300 years ago. The disease is characterized by fiber-like strands extruding from the skin in conjunction with various dermatologic and neuropsychiatric symptoms. In this respect, Morgellons disease resembles and may be confused with delusional parasitosis. The association with Lyme disease and the apparent response to antibacterial therapy suggest that Morgellons disease may be linked to an undefined infectious process. Further clinical and molecular research is needed to unlock the mystery of Morgellons disease.

Publication Types:
PMID: 16489838 [PubMed - indexed for MEDLINE]

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Inactivation of the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi.

Seshu J, Esteve-Gassent MD, Labandeira-Rey M, Kim JH, Trzeciakowski JP, Höök M, Skare JT.

Department of Microbial and Molecular Pathogenesis, Texas A&M University Health Science Center, 407 Reynolds Medical Building, College Station, 77843, USA.

Borrelia burgdorferi, the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surface-exposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi. Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.

Publication Types:
PMID: 16468997 [PubMed - indexed for MEDLINE]

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Neuralgia and demyelinating plaques: MS or lyme disease?

Savely G.

South Austin Family Practice Clinic, Austin, Texas, USA.

Publication Types:
PMID: 16152811 [PubMed - indexed for MEDLINE]

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Primary cutaneous B-cell lymphoma.

Bogle MA, Riddle CC, Triana EM, Jones D, Duvic M.

Department of Dermatology, University of Texas and M. D. Anderson Cancer Center, Houston, Texas 77030, USA. mabogle@hotmail.com

Primary cutaneous B-cell lymphomas include extranodal marginal zone B-cell lymphoma, follicular lymphoma, large B-cell lymphoma, and, rarely, mantle cell lymphoma. Our purpose in conducting this review was to determine the clinical and behavioral characteristics of primary cutaneous B-cell lymphomas, their relationship to infectious triggers, and therapeutic response. We conducted a retrospective chart review of 23 adult patients presenting to the dermatology clinic at M. D. Anderson Cancer Center with primary cutaneous B-cell lymphoma between January 1999 and May 2003. Primary cutaneous B-cell lymphomas generally present on the head and neck, with the trunk and extremities afflicted to a lesser extent. Patients were found to have serologic evidence of prior infection with Borrelia burgdorferi (n = 10), Helicobacter pylori (n = 5), and Epstein-Barr virus (n = 6). Overall, treatment of primary cutaneous B-cell lymphoma should involve multiple modalities; however, specific treatment aimed at concurrent or suspected infection, particularly B burgdorferi, is a helpful adjunct and may achieve complete remission in a small subset of patients.

PMID: 16112357 [PubMed - indexed for MEDLINE]

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Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi.

Yang XF, Lybecker MC, Pal U, Alani SM, Blevins J, Revel AT, Samuels DS, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, 75390-9048, USA.

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.

Publication Types:
PMID: 15995197 [PubMed - indexed for MEDLINE]

PMCID: PMC1169512


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Multicomponent Lyme vaccine: three is not a crowd.

Brown EL, Kim JH, Reisenbichler ES, Höök M.

The Center for Extracellular Matrix Biology, Texas A&M University System Health Science Center, Albert B. Alkek Institute of Biosciences and Technology, 2121 W, Holcombe Blvd., Suite 603, Houston, TX 77030, USA. ebrown2@uh.edu

Lyme disease is caused by the spirochete Borrelia burgdorferi and it is the most common vector-borne disease in the United States. Disseminated spirochetes can persist in various tissues and can result in a variety of different disease manifestations. Vaccination trials testing various lipoprotein candidates have yielded mixed results despite the generation of robust antibody titers. Data presented in this report demonstrate that a combination vaccine composed of DbpA, BBK32 and OspC is more effective than single or double component formulations and that the ratio of each component dramatically impacts vaccine efficacy when tested in protection experiments against Borrelia following needle inoculation.

Publication Types:
PMID: 15882529 [PubMed - indexed for MEDLINE]

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bptA (bbe16) is essential for the persistence of the Lyme disease spirochete, Borrelia burgdorferi, in its natural tick vector.

Revel AT, Blevins JS, Almazán C, Neil L, Kocan KM, de la Fuente J, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Borrelia burgdorferi (Bb), the agent of Lyme disease, is a zoonotic spirochetal bacterium that depends on arthropod (Ixodes ticks) and mammalian (rodent) hosts for its persistence in nature. The quest to identify borrelial genes responsible for Bb's parasitic dependence on these two diverse hosts has been hampered by limitations in the ability to genetically manipulate virulent strains of Bb. Despite this constraint, we report herein the inactivation and genetic complementation of a linear plasmid-25-encoded gene (bbe16) to assess its role in the virulence, pathogenesis, and survival of Bb during its natural life cycle. bbe16 was found to potentiate the virulence of Bb in the murine model of Lyme borreliosis and was essential for the persistence of Bb in Ixodes scapularis ticks. As such, we have renamed bbe16 a gene encoding borrelial persistence in ticks (bpt)A. Although protease accessibility experiments suggested that BptA as a putative lipoprotein is surface-exposed on the outer membrane of Bb, the molecular mechanism(s) by which BptA promotes Bb persistence within its tick vector remains to be elucidated. BptA also was shown to be highly conserved (>88% similarity and >74% identity at the deduced amino acid levels) in all Bb sensu lato strains tested, suggesting that BptA may be widely used by Lyme borreliosis spirochetes for persistence in nature. Given Bb's absolute dependence on and intimate association with its arthropod and mammalian hosts, BptA should be considered a virulence factor critical for Bb's overall parasitic strategy.

Publication Types:
PMID: 15860579 [PubMed - indexed for MEDLINE]

PMCID: PMC1100799


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Immune evasion of the Lyme disease spirochetes.

Bubeck-Martinez S.

Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. martinezsb@uthscsa.edu <martinezsb@uthscsa.edu>

The Lyme disease spirochetes, Borrelia burgdorferi sensu lato, have adapted very well to both surviving and persisting in the mammalian host despite a strong host antibody response. It appears that both temporal and spatial regulation of outer surface proteins have contributed to this persistence. The spirochetes are able to bind fH and FHL-1 to their surface, resulting in decreased complement activation. In addition, the organisms have taken advantage of components of tick saliva to aid in their initial immune evasion and dissemination. Studies leading to these conclusions are reviewed here.

Publication Types:
PMID: 15569625 [PubMed - indexed for MEDLINE]

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A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi.

Seshu J, Boylan JA, Hyde JA, Swingle KL, Gherardini FC, Skare JT.

Department of Medical Microbiology and Immunology, 407 Reynolds Medical Building, Texas A&M University Health Science Center, College Station, TX 77843, USA.

Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi. When we tested the sensitivity of B. burgdorferi to ROS, one clonal non-infectious B. burgdorferi isolate exhibited hypersensitivity to t-butyl hydroperoxide when compared with infectious B. burgdorferi and other non-infectious isolates. Sequence analysis indicated that the hypersensitive non-infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non-infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t-butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter-lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA). In addition, we determined that cells lacking BosRR39K synthesized fourfold greater levels of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.

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PMID: 15554974 [PubMed - indexed for MEDLINE]

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Effects of vlsE complementation on the infectivity of Borrelia burgdorferi lacking the linear plasmid lp28-1.

Lawrenz MB, Wooten RM, Norris SJ.

Department of Pathology and Laboratory Medicine, Graduate School of Biomedical Sciences, University of Texas--Houston Health Science Center, USA.

The loss of linear plasmid lp28-1, which contains the vls antigenic variation locus, is associated with reduced infectivity of Borrelia burgdorferi in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable B. burgdorferi lacking lp25 and lp56, was used to determine the effect of trans expression of vlsE on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and vlsE. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured B. burgdorferi, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact vls antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the vlsE shuttle plasmid and was examined for vlsE sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion. Lack of vlsE sequence variation on the shuttle plasmid thus did not result in clearance of the trans-complemented strain in immunocompetent mice under the conditions tested.

Publication Types:
PMID: 15501789 [PubMed - indexed for MEDLINE]

PMCID: PMC523020


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BBK32, a fibronectin binding MSCRAMM from Borrelia burgdorferi, contains a disordered region that undergoes a conformational change on ligand binding.

Kim JH, Singvall J, Schwarz-Linek U, Johnson BJ, Potts JR, Höök M.

Center for Extracellular Matrix Biology, Albert B. Alkek Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Houston, Texas 77030, USA.

BBK32 is a fibronectin-binding lipoprotein on Borrelia burgdorferi, the causative agent of Lyme disease. Analysis using secondary structure prediction programs suggested that BBK32 is composed of two domains, an N-terminal segment lacking well defined secondary structure and a C-terminal segment composed largely of alpha-helices. Analysis of purified recombinant forms of the two domains by circular dichroism spectroscopy, gel permeation chromatography, and intrinsic viscosity determination were consistent with an N-terminal-extended, unstructured segment and a C-terminal globular domain in BBK32. Solid phase binding experiments suggest that the unstructured N-terminal domain binds fibronectin. Analysis of changes in circular dichroism spectra of the N-terminal segment of BBK32 upon binding of the N-terminal domain of fibronectin revealed an increase in beta-sheet content in the complex. Hence, BBK32, which belongs to a different family of proteins and shows no overall sequence similarity with the fibronectin binding MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) of Gram-positive bacteria, binds fibronectin by a mechanism that is reminiscent of the "tandem beta-zipper" previously demonstrated for the fibronectin binding of streptococcal adhesins.

Publication Types:
PMID: 15292204 [PubMed - indexed for MEDLINE]

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The luxS gene is not required for Borrelia burgdorferi tick colonization, transmission to a mammalian host, or induction of disease.

Blevins JS, Revel AT, Caimano MJ, Yang XF, Richardson JA, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, 75390, USA.

luxS mutants of Borrelia burgdorferi strain 297 naturally colonized their arthropod (Ixodes scapularis) vector, were maintained in ticks throughout the molting process (larvae to nymphs), were tick transmitted to uninfected mice, and elicited histopathology in mice indistinguishable from that induced by wild-type B. burgdorferi.

Publication Types:
PMID: 15271949 [PubMed - indexed for MEDLINE]

PMCID: PMC470628


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Erratum in:
  • Infect Immun. 2004 Apr;72(4):2456.

Dissolved oxygen levels alter gene expression and antigen profiles in Borrelia burgdorferi.

Seshu J, Boylan JA, Gherardini FC, Skare JT.

Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.

The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in B. burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B. burgdorferi was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in B. burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

Publication Types:
PMID: 14977964 [PubMed - indexed for MEDLINE]

PMCID: PMC356058


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The response regulator Rrp2 is essential for the expression of major membrane lipoproteins in Borrelia burgdorferi.

Yang XF, Alani SM, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.

Borrelia burgdorferi (Bb), the agent of Lyme disease, exists in nature through a complex enzootic life cycle that involves both ticks and mammals. As Bb transitions between its two diverse niches, profound adaptive changes occur that are reflected in differential patterns of gene expression, particularly involving lipoprotein genes. Using a mutagenesis approach, we show that Rrp2 (gene BB0763), one of the proteins predicted by the Bb genome (www.tigr.org) to be a response regulator of a two-component sensory transduction system, is a pivotal regulator governing the expression of major membrane lipoproteins such as OspC, DbpA, and Mlp8, as well as many other mammalian infection-associated immunogens of Bb. Sequence analysis additionally suggested that Rrp2 is a bacterial enhancer-binding protein, essential for sigma54-dependent gene activation. Mutagenesis of a key amino acid residue within a putative activation domain revealed that Rrp2 controlled lipoprotein expression by governing the expression of the alternative sigma-factor sigmas in a sigma54-dependent manner. We therefore propose a signal transduction pathway involving Rrp2, sigma54, and sigmas, which in concert control the expression of key lipoproteins and other infection-associated immunogens in Bb.

Publication Types:
PMID: 12949258 [PubMed - indexed for MEDLINE]

PMCID: PMC196916


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Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi.

Yang XF, Hübner A, Popova TG, Hagman KE, Norgard MV.

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

The Mlp (multicopy lipoproteins) family is one of many paralogous protein families in Borrelia burgdorferi. To examine the extent to which the 10 members of the Mlp family in B. burgdorferi strain 297 might be differentially regulated, antibodies specific for each of the Mlps were developed and used to analyze the protein expression profiles of individual Mlps when B. burgdorferi replicated under various cultivation conditions. All of the Mlps were upregulated coordinately when B. burgdorferi was cultivated at either elevated temperature, reduced culture pH, or increased spirochete cell density. Inasmuch as the expression of OspC is influenced by a novel RpoN-RpoS regulatory pathway, similar induction patterns for OspC and the Mlp paralogs prompted an assessment of whether the RpoN-RpoS pathway also was involved in Mlp expression. In contrast to wild-type B. burgdorferi, both RpoN- and RpoS-deficient mutants were unable to upregulate OspC or the Mlp paralogs when grown at lower pH (6.8), indicating that pH-mediated regulation of OspC and Mlp paralogs is dependent on the RpoN-RpoS pathway. However, when RpoN- or RpoS-deficient mutants were shifted from 23 degrees C to 37 degrees C or were cultivated to higher spirochete densities, some induction of the Mlps still occurred, whereas OspC expression was abolished. The combined findings suggest that the Mlp paralogs are coordinately regulated as a family and have an expression profile similar, but not identical, to that of OspC. Although Mlp expression as a family is influenced by the RpoN-RpoS regulatory pathway, there exists at least one additional layer of gene regulation, yet to be elucidated, contributing to Mlp expression in B. burgdorferi.

Publication Types:
PMID: 12933844 [PubMed - indexed for MEDLINE]

PMCID: PMC187337


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The absence of linear plasmid 25 or 28-1 of Borrelia burgdorferi dramatically alters the kinetics of experimental infection via distinct mechanisms.

Labandeira-Rey M, Seshu J, Skare JT.

Department of Medical Microbiology and Immunology, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.

The 25-kb linear plasmid lp25 and one of the 28-kb linear plasmids (lp28-1) are required for experimental infection in Borrelia burgdorferi, the etiologic agent of Lyme disease. The loss of these plasmids either eliminates infectivity (lp25) or significantly increases the 50% infective dose during a 2-week infection period (lp28-1). This study assessed the kinetics of bacterial dissemination in C3H/HeN mice infected with B. burgdorferi lacking either lp25 or lp28-1, as well as their wild-type parent, and tracked the development of specific borrelial antibodies over a 3-week period. The results indicated that the wild type and the lp28-1(-) strains were able to disseminate throughout the host, whereas the lp25(-) strain was cleared within 48 h of inoculation. While the wild-type B. burgdorferi persisted in tissues for the duration of the study, the lp28-1(-) mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1(-) mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1(-) mutant was able to disseminate and persist in C3H-scid mice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis.

Publication Types:
PMID: 12874340 [PubMed - indexed for MEDLINE]

PMCID: PMC166013


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Effect of complement component C3 deficiency on experimental Lyme borreliosis in mice.

Lawrenz MB, Wooten RM, Zachary JF, Drouin SM, Weis JJ, Wetsel RA, Norris SJ.

Program in Microbiology and Molecular Genetics and Department of Pathology and Laboratory Medicine, Graduate School of Biomedical Sciences, University of Texas-Houston Health Science Center, Houston, Texas, USA.

Mice deficient in complement component C3 (C3(-/-)) and syngeneic C57BL/6 control mice were challenged with Borrelia burgdorferi to determine the role of complement in immune clearance and joint histopathology during experimental Lyme borreliosis. Tibiotarsal joint, ear, and heart tissues were monitored for spirochete numbers at 2, 4, 8, and 12 weeks postinoculation with 10(5) B. burgdorferi B31 clone 5A4 by using quantitative real-time PCR. The spirochete load in joint and ear tissue remained higher in the C3(-/-) mice than in the wild-type counterparts throughout the 12-week study, whereas the numbers in heart tissue of both groups of mice decreased substantially at 8 to 12 weeks postinfection. Histopathology scores for joint tissue were generally higher in the C3(-/-) mice compared to C57BL/6 controls at 2 and 4 weeks postinfection, which may reflect the presence of higher numbers of bacteria in the joints at these early time points. Levels of anti-B. burgdorferi immunoglobulin G tended to be reduced in the C3(-/-) mice compared to control mice. Furthermore, a 5.5-fold-lower number of the complement-sensitive Borrelia garinii was needed to infect C3(-/-) mice compared to C57BL/6 mice, indicating that its sensitivity to complement is one barrier to infection of the mouse model by B. garinii. These results indicate that the complement system may be important in controlling the early dissemination and progression of B. burgdorferi infection.

Publication Types:
PMID: 12874322 [PubMed - indexed for MEDLINE]

PMCID: PMC165993


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