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PubMed1999

Characterization of VspB of Borrelia turicatae, a major outer membrane protein expressed in blood and tissues of mice.

Serotypes A and B of the relapsing fever spirochete Borrelia turicatae produce different disease manifestations in infected mice. Whereas serotype B causes more severe arthritis and reaches higher densities in the blood of mice than serotype A, serotype A invades the central nervous system earlier than serotype B during infection. These differences between serotypes A and B in mice are associated with the expression of different surface proteins, VspA and VspB, respectively, in the culture medium. To determine whether these proteins, in particular, VspB, are also expressed in vivo, scid mice infected with B. turicatae were studied. The expression of VspB by spirochetes in the blood was demonstrated in Coomassie blue-stained polyacrylamide gels and Western blots with a specific monoclonal antibody. Indirect immunofluorescence and immunoperoxidase studies confirmed the expression of VspB in the blood and also demonstrated VspB expression in the joints and heart. The gene for VspB was next identified and cloned by using partial amino acid sequencing, reverse transcriptase PCR, and a specific monoclonal antibody. The vspB gene encodes a protein of 216 amino acids that is 68% identical to VspA of B. turicatae and 44 to 56% identical to representative Vsp and OspC lipoproteins of other Borrelia spp. The processed VspB protein was distinguished from 26 other Vsp and OspC proteins by a high predicted isoelectric point at 9.39. The promoter region for vspB was similar to the promoter region for the vsp33 gene of Borrelia hermsii and for the ospC gene of Borrelia burgdorferi, two genes known to be environmentally regulated. These studies established that the virulence-associated VspB protein is expressed by spirochetes in the mouse and that VspB is a novel member of the Vsp-OspC family of proteins.

Amino Acid Sequence↗
PubMed2000

Conservation and heterogeneity of vlsE among human and tick isolates of Borrelia burgdorferi.

The vls (variable major protein [VMP]-like sequence) locus of Borrelia burgdorferi encodes an antigenic variation system that closely resembles the VMP system of relapsing fever borreliae. To determine whether vls sequences are present consistently in low-passage, infectious isolates of B. burgdorferi, 22 blood and erythema migrans biopsy isolates from Lyme disease patients in Westchester County, New York, were examined by Southern blot and PCR analysis. Each of the strains contained a single plasmid varying in size from 21 to 38 kb that hybridized strongly with a vlsE probe based on the B. burgdorferi B31 sequence. In contrast, PCR products were obtained with only 10 of the 22 strains when primers corresponding to the 5' and 3' regions of the B31 vlsE sequence outside the variable cassette region were used. Only 2 of 16 B. burgdorferi-infected tick specimens yielded detectable PCR product. Eight of 10 strains that yielded a PCR product under these conditions were type 1 (a genotype with a high rate of dissemination), according to PCR-restriction fragment length polymorphism analysis of intergenic rDNA sequences, whereas the isolates that did not yield vlsE PCR products were either type 2 or type 3. Comparison of the sequences of cloned PCR products from the patient isolates indicated a high degree of identity to the B31 sequence, with most of the differences restricted to the hypervariable regions known to undergo sequence variation. Taken together, these results both reinforce previous evidence that vls sequences are present consistently in low-passage Lyme disease spirochetes and indicate that both highly conserved and heterogeneous subgroups exist with regard to vlsE sequences.

Amino Acid Sequence↗
PubMed2000

Direct evidence for involvement of NF-kappaB in transcriptional activation of tumor necrosis factor by a spirochetal lipoprotein.

Variable major lipoprotein (Vmp) is a major tumor necrosis factor (TNF)-inducing component of Borrelia recurrentis, the agent of louse-borne relapsing fever. B. recurrentis Vmp rapidly stimulates nuclear translocation of NF-kappaB and proinflammatory cytokine gene expression in the human monocyte-like cell line MonoMac 6. By overexpressing disabled mutant IkappaBalpha in MonoMac 6 cells cotransfected with a reporter gene, we provide evidence that NF-kappaB is essential for the transcriptional activation of TNF in this system.

Bacterial Proteins↗
PubMed2001

Borrelia spirochetes upregulate release and activation of matrix metalloproteinase gelatinase B (MMP-9) and collagenase 1 (MMP-1) in human cells.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, stimulated human peripheral blood monocytes to release pro-matrix metalloproteinase-9 (gelatinase B; pro-MMP-9) and active matrix metalloproteinase-1 (collagenase-1; MMP-1). Human neutrophils also released pro-MMP-9 and a 130-kDa protein with gelatinolytic activity in response to live B. burgdorferi. In addition, U937 cells and human keratinocyte cells were also stimulated to release pro-MMP-9 under the same conditions. However, human umbilical vein endothelial cells (HUVECs) released pro-MMP-9 and pro-MMP-2 in a constitutive manner and were not influenced by live spirochetes. MMPs produced by human monocytes also enhanced the penetration of B. burgdorferi through extracellular matrix component barriers in vitro. Plasmin stabilized on the surface of the Lyme disease spirochete was shown to activate pro-MMP-9 to its active form. This active form was also observed in the plasma of mice infected with a relapsing fever borrelia. These results suggest that borreliae can upregulate MMPs and possibly mediate an activation cascade initiated by plasmin bound to the microbial surface. MMPs may play a role in dissemination of the Lyme disease spirochete and in the pathogenesis of Borrelia infection.

Animals↗
PubMed2001

Isogenic serotypes of Borrelia turicatae show different localization in the brain and skin of mice.

Mice with severe combined immunodeficiency (scid mice) and infected with the relapsing fever agent Borrelia turicatae develop manifestations that resemble those of disseminated Lyme disease. We have characterized two isogenic serotypes, A and B, which differ in their variable small proteins (Vsps) and disease manifestations. Serotype A but not serotype B was cultured from the brain during early infection, and serotype B caused more severe arthritis, myocarditis, and vestibular dysfunction than serotype A. Here we compared the localization and number of spirochetes and the severity of inflammation in scid mice, using immunostained and hematoxylin-and-eosin-stained coronal sections of decalcified heads. Spirochetes in the brain localized predominantly to the leptomeninges, and those in peripheral tissues localized mainly to the extracellular matrix. There were significantly more serotype A than B spirochetes in the leptomeninges and more serotype B than A spirochetes in the skin. The first tissue where spirochetes were observed outside the vasculature was the dura mater. Inflammation was more severe in the skin than in the brain. VspA, VspB, and the periplasmic flagellin protein were expressed in all tissues examined. These findings indicate that isogenic but antigenically distinct Borrelia serotypes can have marked differences in their localization in tissues.

Animals↗
PubMed2001

Delayed invasion of the kidney and brain by Borrelia crocidurae in plasminogen-deficient mice.

Borrelia crocidurae is an etiologic agent of relapsing fever in Africa and is transmitted to humans by the bite of soft ticks of the genus Ornithodoros. The role of the plasminogen (Plg) activation system for the pathogenicity of B. crocidurae was investigated by infection of Plg-deficient (plg(-/-)) and Plg wild-type (plg(+/+)) mice. No differences in spirochetemia were observed between the plg(-/-) and plg(+/+) mice. However, signs indicative of brain invasion, such as neurological symptoms and/or histopathological changes, were more common in plg(+/+) mice. Quantitative immunohistochemical analysis demonstrated infection of spirochetes in kidney interstitium and brain as soon as 2 days postinoculation. Lower numbers of extravascular spirochetes in plg(-/-) mice during the first days of infection suggested a less efficient invasion mechanism in these mice than in the plg(+/+) mice. The invasion of the kidneys in plg(-/-) mice produced no significant inflammation, as seen by quantitative immunohistochemistry of the CD45 common leukocyte marker. However, significant kidney inflammation was observed with infection in the plg(+/+) mice. In brain, inflammation was more severe in plg(+/+) mice than in plg(-/-) mice, and the numbers of CD45(+) cells increased significantly with duration of infection in the plg(+/+) mice. The results show that invasion of brain and kidney occurs as early as 2 days after inoculation. Also, Plg is not required for establishment of spirochetemia by the organism, whereas it is involved in the invasion of organs.

Animals↗
PubMed2004

Cross-species surface display of functional spirochetal lipoproteins by recombinant Borrelia burgdorferi.

Surface-exposed lipoproteins of relapsing fever (RF) and Lyme borreliosis Borrelia spirochetes mediate certain interactions of the bacteria with their arthropod and vertebrate hosts. RF spirochetes such as Borrelia hermsii serially evade the host's antibody response by multiphasic antigenic variation of Vsp and Vlp proteins. Furthermore, the expression of Vsp1 and Vsp2 by Borrelia turicatae is associated with neurotropism and higher blood densities, respectively. In contrast to RF Borrelia species, the Lyme borreliosis spirochete Borrelia burgdorferi is amenable to genetic manipulation. To facilitate structure-function analyses of RF surface lipoproteins, we used recombinant plasmids to introduce full-length vsp1 and vsp2 as well as two representative vlp genes into B. burgdorferi cells. Recombinant B. burgdorferi cells constitutively expressed the proteins under the control of the B. burgdorferi flaB promoter. Antibody and protease accessibility assays indicated proper surface exposure and folding. Expression of Vsp1 and Vsp2 conferred glycosaminoglycan binding to recombinant B. burgdorferi cells that was similar to that observed with purified recombinant proteins and B. turicatae expressing native Vsp. These data demonstrate that the lipoprotein modification and export mechanisms in the genus Borrelia are conserved. They also validate the use of recombinant B. burgdorferi in studies of surface lipoprotein structure-function and the biogenesis of spirochete membranes.

Animals↗
PubMed2006

Differential telomere processing by Borrelia telomere resolvases in vitro but not in vivo.

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins.

Amino Acid Sequence↗
PubMed1990

Methylated DNA in Borrelia species.

The DNA of Borrelia species was examined for the presence of methylated GATC sequences. The relapsing-fever Borrelia sp., B. coriaceae, and only 3 of 22 strains of B. burgdorferi contained adenine methylation systems. B. anserina lacked an adenine methylation system. Fundamental differences in DNA methylation exist among members of the genus Borrelia.

Base Sequence↗
PubMed1994

Plasmid location of Borrelia purine biosynthesis gene homologs.

The Lyme disease spirochete Borrelia burgdorferi must survive in both its tick vector and its mammalian host to be maintained in nature. We have identified the B. burgdorferi guaA gene encoding GMP synthetase, an enzyme involved in de novo purine biosynthesis that is important for the survival of bacteria in mammalian blood. This gene encodes a functional product that will complement an Escherichia coli GMP synthetase mutant. The gene is located on a 26-kb circular plasmid, adjacent to and divergent from the gene encoding the outer surface protein C (OspC). The guaB gene homolog encoding IMP dehydrogenase, another enzyme in the purine biosynthetic pathway, is adjacent to guaA. In Borrelia hermsii, a tick-borne relapsing fever spirochete, the guaA and guaB genes are located on a linear plasmid. These are the first genes encoding proteins of known function to be mapped to a borrelial plasmid and the only example of genes encoding enzymes involved in the de novo purine biosynthesis pathway to be mapped to a plasmid in any organism. The unique plasmid location of these and perhaps other housekeeping genes may be a consequence of the segmented genomes in borreliae and reflect the need to adapt to both the arthropod and mammalian environments.

Amino Acid Sequence↗
PubMed1997

The bacterial nucleoid visualized by fluorescence microscopy of cells lysed within agarose: comparison of Escherichia coli and spirochetes of the genus Borrelia.

The nucleoids of Escherichia coli and the spirochetes Borrelia burgdorferi and Borrelia hermsii, agents of Lyme disease and relapsing fever, were examined by epifluorescence microscopy of bacterial cells embedded in agarose and lysed in situ with detergent and protease. The typical E. coli nucleoid was a rosette in which 20 to 50 long loops of DNA emanated from a dense node of DNA. The percentages of cells in a population having nucleoids with zero, one, two, and three nodes varied with growth rate and growth phase. The borrelia nucleoid, in contrast, was a loose network of DNA strands devoid of nodes. This nucleoid structure difference correlates with the unusual genome of Borrelia species, which consists primarily of linear replicons, including a 950-kb linear chromosome and linear plasmids. This method provides a simple, direct means to analyze the structure of the bacterial nucleoid.

Borrelia↗
PubMed2000

Analysis of the cellular localization of Bdr paralogs in Borrelia burgdorferi, a causative agent of lyme disease: evidence for functional diversity.

The bdr (Borrelia direct repeat) gene family of the genus Borrelia encodes a polymorphic group of proteins that carry a central repeat motif region containing putative phosphorylation sites and a hydrophobic carboxyl-terminal domain. It has been postulated that the Bdr proteins may anchor to the inner membrane via the C-terminal domain. In this study, we used cellular fractionation methodologies, salt and detergent treatments, and immunoblot analyses to assess the association of the Bdr proteins with the cellular infrastructure in both Borrelia burgdorferi (a Lyme disease spirochete) and B. turicatae (a relapsing fever spirochete). Triton X-114 extraction and partitioning experiments demonstrated that most Bdr paralogs are associated with the inner membrane-peptidoglycan complex. Analyses of cells treated with the highly chaotropic bile salt detergent deoxycholic acid demonstrated that some Bdr paralogs may also interact with the peptidoglycan, as evidenced by their tight association with the insoluble cellular matrix. In addition, immunoprecipitation (IP) experiments revealed an enhanced IP of all Bdr paralogs when the cell lysates were boiled prior to addition of the precipitating antibody. Furthermore, some Bdr paralogs were accessible to antibody in the IP experiments only in the boiled cell lysates. These observations suggest that different Bdr paralogs may carry out different structural-functional roles. Demonstration of the inner membrane localization of the Bdr proteins and of the differences in nature of the interaction of individual Bdr paralogs with the cell infrastructure is an important step toward defining the functional role of this unique protein family in the genus Borrelia.

Bacterial Proteins↗
PubMed2004

Transgenic expression of RecA of the spirochetes Borrelia burgdorferi and Borrelia hermsii in Escherichia coli revealed differences in DNA repair and recombination phenotypes.

After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B. burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli. As a control, E. coli RecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage lambda. Overall, we found that transgenic cells with recA genes of B. burgdorferi, B. hermsii, and L. biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B. burgdorferi recA and, most notably, B. hermsii recA were significantly less capable than cells with L. biflexa recA or E. coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E. coli.

Amino Acid Sequence↗
PubMed2005

Putative coiled-coil structural elements of the BBA68 protein of Lyme disease spirochetes are required for formation of its factor H binding site.

Factor H and factor H like-protein 1 (FHL-1) are complement regulatory proteins that serve as cofactors for the factor I-mediated cleavage of C3b. Some Lyme disease and relapsing fever spirochete species bind factor H to their surface to facilitate immune evasion. The Lyme disease spirochetes produce several factor H binding proteins (FHBPs) that form two distinct classes. Class I FHBPs (OspE orthologs and paralogs) bind only factor H, while class II FHBPs (BBA68) bind both factor H and FHL-1. BBA68 belongs to a large paralogous protein family, and of these paralogs, BBA69 is the member most closely related to BBA68. To determine if BBA69 can also bind factor H, recombinant protein was generated and tested for factor H binding. BBA69 did not exhibit factor H binding ability, suggesting that among family 54 paralogs, factor H binding is unique to BBA68. To identify the determinants of BBA68 that are involved in factor H binding, truncation and site-directed mutational analyses were performed. These analyses revealed that the factor H binding site is discontinuous and provide strong evidence that coiled-coil structural elements are involved in the formation of the binding site.

Amino Acid Sequence↗
PubMed2006

Function and evolution of plasmid-borne genes for pyrimidine biosynthesis in Borrelia spp.

The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.

Amino Acid Sequence↗
PubMed1990

Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi.

Borrelia burgdorferi is the causative agent of Lyme borreliosis, a spirochetal illness with a variety of acute clinical manifestations that may lead to debilitating neurological and arthritic complications. Diagnosis is difficult because symptoms mimic a variety of unrelated clinical conditions, spirochetes cannot always be isolated from infected patients, and current serological tests are frequently inconclusive because of the presence of cross-reacting non-B. burgdorferi antibodies. To identify antigens specific to B. burgdorferi that could be used in the serodiagnosis of Lyme borreliosis, we screened a Borrelia DNA expression library in Escherichia coli for antigens reactive with human Lyme borreliosis sera. One clone carried a 6.3-kilobase EcoRI chromosomal fragment (pSPR33), which encoded two species-specific antigens with molecular masses of 28 (P28) and 39 (P39) kilodaltons (kDa). These two antigens were immunologically distinct from OspA, OspB, and the 41-kDa flagellin. Ninety-four serum specimens from patients having Lyme borreliosis were tested for reactivity with P39. All of 33 the serum specimens with immunofluorescence assay titers of greater than or equal to 1:256, 13 of 17 serum specimens with titers of 1:128, and 14 of 44 serum specimens with titers of less than or equal to 1:64 reacted with P39. Notably, many sera reactive to P39 did not appear to react with the 41-kDa flagellin. Therefore, antibody to P39 could be mistaken for antibody to the 41-kDa flagellin in tests of human sera by Western blot (immunoblot). Twenty-five control serum specimens, which included sera from syphilitic, relapsing fever, and amyotrophic lateral sclerosis patients as well as from 10 normal individuals, did not react to P39. Our data suggest that P39 may be a useful antigen for the serological confirmation of Lyme borreliosis.

Animals↗
PubMed1991

Polymerase chain reaction analyses identify two distinct classes of Borrelia burgdorferi.

We sequenced homologous chromosomal loci from several North American and European isolates of the Lyme disease spirochete Borrelia burgdorferi, as well as from the relapsing fever spirochete Borrelia hermsii. Inter- and intraspecies sequence comparisons permitted the design of B. burgdorferi-specific polymerase chain reaction primers that detected all strains tested (n = 31) from diverse geographical and biological origins. Polymerase chain reaction "typing" with other unique sets of primers subdivided B. burgdorferi isolates into two groups: all North American isolates and a few European isolates made up one group, while the majority of the European and Asian isolates made up the second group. This classification may have a clinical correlate reflected in differences between "typical" Lyme borreliosis in North America and Europe.

Animals↗
PubMed1991

Lyme disease assay which detects killed Borrelia burgdorferi.

We developed an in vitro assay showing that Borrelia burgdorferi organisms were killed by serum from patients with Lyme disease. Twenty of 20 Lyme disease serum samples caused B. burgdorferi killing in a range of 36 to 99% compared with the mean number of viable spirochetes when sera from 10 healthy individuals were used. The percentage of killing of B. burgdorferi increased with convalescent serum from patients with early Lyme disease. The borreliacidal activity was detectable in some sera diluted 640-fold and was abrogated after treatment with anti-human immunoglobulin G. In contrast, pooled or individual normal human serum did not cause a decrease in the number of viable B. burgdorferi. Borreliacidal activity was also not detected in sera from patients with relapsing fever, rocky mountain spotted fever, syphilis, mononucleosis, rheumatoid factor, or DNA antibodies. Our results show that borreliacidal activity can be used as a specific serodiagnostic test for detecting Lyme disease.

Antibodies, Bacterial↗