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Interactions among pathogen genotypes that vary in host specificity may affect overall transmission dynamics in multi-host systems. Borrelia burgdorferi , a bacterium that causes Lyme disease, is typically transmitted among wildlife by Ixodes ticks. Despite the existence of many alleles of B. burgdorferi 's sensu stricto outer surface protein C ( ospC ) gene, most human infections are caused by a small number of ospC alleles ["human infectious alleles" (HIAs)], suggesting variation in host specificity associated with ospC . To characterize the wildlife host association of B. burgdorferi 's ospC alleles, we used metagenomics to sequence ospC alleles from 68 infected individuals belonging to eight mammalian species trapped at three sites in suburban New Brunswick, New Jersey (USA). We found that multiple allele ("mixed") infections were common. HIAs were most common in mice ( Peromyscus spp.) and only one HIA was detected at a site where mice were rarely captured. ospC allele U was exclusively found in chipmunks ( Tamias striatus ), and although a significant number of different alleles were observed in chipmunks, including HIAs, allele U never co-occurred with other alleles in mixed infections. Our results suggest that allele U may be excluding other alleles, thereby reducing the capacity of chipmunks to act as reservoirs for HIAs., Competing Interests: The authors declare no conflict of interest.

Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site. If hematogenous dissemination occurs, the patient may then develop neurologic manifestations, carditis, or arthritis. Host-pathogen interactions include factors that contribute to hematogenous dissemination to other body sites. Outer surface protein C (OspC), a surface-exposed lipoprotein of B. burgdorferi, is essential during the early stages of mammalian infection. There is a high degree of genetic variation at the ospC locus, and certain ospC types are more frequently associated with hematogenous dissemination in patients, suggesting that OspC may be a major contributing factor to the clinical outcome of B. burgdorferi infection. In order to evaluate the role of OspC in B. burgdorferi dissemination, ospC was exchanged between B. burgdorferi isolates with different capacities to disseminate in laboratory mice, and these strains were then tested for their ability to disseminate in mice. The results indicated that the ability of B. burgdorferi to disseminate in mammalian hosts does not depend on OspC alone. The complete genome sequences of two closely related strains of B. burgdorferi with differing dissemination phenotypes were determined, but a specific genetic locus that could explain the differences in the phenotypes could not be definitively identified. The animal studies performed clearly demonstrated that OspC is not the sole determinant of dissemination. Future studies of the type described here with additional borrelial strains will hopefully clarify the genetic elements associated with hematogenous dissemination.

The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, development might be restricted either to conserved antigens, which are often rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the United States and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions, based primarily on binding sites of monoclonal antibodies (MAbs). In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed, and, based on their immunogenicity, four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4) or with in vitro -grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro -grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis., (Copyright © 2020 American Society for Microbiology.)

The global public health impact of relapsing fever (RF) spirochetosis is significant, since the pathogens exist on five of seven continents. The hallmark sign of infection is episodic fever and the greatest threat is to the unborn. With the goal of better understanding the specificity of B-cell responses and the role of immune responses in pathogenicity, we infected rhesus macaques with Borrelia turicatae (a new world RF spirochete species) by tick bite and monitored the immune responses generated in response to the pathogen. Specifically, we evaluated inflammatory mediator induction by the pathogen, host antibody responses to specific antigens, and peripheral lymphocyte population dynamics. Our results indicate that B. turicatae elicits from peripheral blood cells key inflammatory response mediators (interleukin-1β and tumor necrosis factor alpha), which are associated with preterm abortion. Moreover, a global decline in peripheral B-cell populations was observed in all animals at 14 days postinfection. Serological responses were also evaluated to assess the antigenicity of three surface proteins: BipA, BrpA, and Bta112. Interestingly, a distinction was observed between antibodies generated in nonhuman primates and mice. Our results provide support for the nonhuman primate model not only in studies of prenatal pathogenesis but also for diagnostic and vaccine antigen identification and testing., (Copyright © 2019 American Society for Microbiology.)

The primary causative agent of tick-borne relapsing fever in North America is Borrelia hermsii. It has been hypothesized that B. hermsii evades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement. In vitro, B. hermsii produces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen for B. hermsii infection in humans. The ability to test the hypothesis that FhbA is a critical virulence factor in vivo has been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated a B. hermsii fhbA deletion mutant (the B. hermsii YORΔfhbA strain) through allelic exchange mutagenesis. Deletion of fhbA abolished FH binding by the YORΔfhbA strain and eliminated cleavage of C3b on the cell surface. However, the YORΔfhbA strain remained infectious in mice and retained resistance to killing in vitro by human complement. Collectively, these results indicate that B. hermsii employs an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Multilocus sequence typing of Borrelia hermsii isolates reveals its divergence into two major genomic groups (GG), but no differences in transmission efficiency or host pathogenicity are associated with these genotypes. To compare GGI and GGII in the tick-host infection cycle, we first determined if spirochetes from the two groups could superinfect the tick vector Ornithodoros hermsi. We infected mice with isolates from each group and fed ticks sequentially on these mice. We then fed the infected ticks on naive mice and measured GGI and GGII spirochete densities in vector and host, using quantitative PCR of genotype-specific chromosomal DNA sequences. Sequential feedings resulted in dual tick infections, showing that GGI or GGII primary acquisition did not block superinfection by a secondary agent. On transmission to naive mice at short intervals after acquisition, ticks with primary GGI and secondary GGII spirochete infections caused mixed GGI and GGII infections in mice. However, ticks with primary GGII and secondary GGI spirochete infections caused only GGII infections with all isolate pairs examined. At longer intervals after acquisition, the exclusion of GGI by GGII spirochetes declined and cotransmission predominated. We then examined GGI and GGII spirochetemia in mice following single inoculation and coinoculation by needle and found that GGI spirochete densities were reduced on multiple days when coinoculated with GGII. These findings indicate that dual GGI-GGII spirochete infections can persist in ticks and that transmission to a vertebrate host is dependent on the order of tick acquisition and the interval between acquisition and transmission events.

Spirochetes belonging to the Borrelia burgdorferi sensu lato complex differ in resistance to complement-mediated killing by human serum. Here, we characterize complement sensitivity of a panel of B. lusitaniae isolates derived from ticks collected in Germany and Portugal as well as one patient-derived isolate, PoHL. All isolates are highly susceptible to complement-mediated lysis in human serum and activate complement predominantly by the alternative pathway, leading to an increased deposition of complement components C3, C6, and the terminal complement complex. Interestingly, serum-sensitive B. lusitaniae isolates were able to bind immune regulator factor H (CFH), and some strains also bound CFH-related protein 1 (CFHR1) and CFHR2. Moreover, CFH bound to the surface of B. lusitaniae was inefficient in mediating C3b conversion. Furthermore, the identification and characterization of a potential CFH-binding protein, OspE, revealed that this molecule possesses a significantly reduced binding capacity for CFH compared to that of CFH-binding OspE paralogs expressed by various serum-resistant Borrelia species. This finding suggests that a reduced binding capability of CFH is associated with an increased serum sensitivity of B. lusitaniae to human complement.

In North America, tick-borne relapsing fever is caused by the species Borrelia hermsii, B. parkeri, and B. turicatae, which are transmitted to humans through the bite of the respective infected tick vectors. Here we describe the identification and functional characterization of a surface lipoprotein of B. parkeri, designated BpcA, that binds the human complement regulators factor H and factor H-related protein 1 and, simultaneously, the host protease plasminogen. In contrast, the homologous B. turicatae protein failed to bind human factor H and factor H-related protein 1 but retained its plasminogen binding capacity. Factor H bound to BpcA maintains its regulatory capacity to control C3b deposition and C3 convertase activity. Ectopic expression of BpcA in a serum-sensitive B. burgdorferi strain protects transformed cells from complement-mediated killing. Furthermore, bound plasminogen/plasmin endows B. parkeri and B. turicatae with the potential to degrade extracellular matrix components. These findings expand our understanding of the putative recent evolutionary separation of Borrelia parkeri and Borrelia turicatae, provide evidence that B. parkeri differs from B. turicatae in its ability to resist complement attack, and may help in understanding the pathological processes underlying tick-borne relapsing fever.

Borrelia hermsii, an etiological agent of tick-borne relapsing fever in North America, binds host-derived serum proteins including factor H (FH), plasminogen, and an unidentified 60-kDa protein via its FhbA protein. Two distinct phylogenetic types of FhbA have been delineated (FhbA1 and FhbA2). These orthologs share a conserved C-terminal domain that contains two alpha helices with a high predictive probability of coiled-coil formation that are separated by a 14-amino-acid loop domain. Through site-directed mutagenesis, we have identified residues within these domains that influence the binding of both mouse and human FH, plasminogen, and/or the 60-kDa protein. To further investigate the involvement of FhbA in the host-pathogen interaction, strains that are either FhbA(+) (isolate YOR) or FhbA(-) (isolate REN) were tested for serum sensitivity. Significant differences were observed, with YOR and REN being serum resistant and serum sensitive (intermediate), respectively. To test the abilities of these strains to infect and persist in mice, mice were needle inoculated, and infectivity and persistence were then assessed. While both strains REN and YOR infected mice, only the FhbA(+) YOR strain persisted beyond day 4. Survival of the YOR isolate in blood correlated with the upregulation of the fhbA gene, as demonstrated by real-time reverse transcriptase PCR. These data advance our understanding of the unique interactions of FhbA with individual serum proteins and provide support for the hypothesis that FhbA is an important contributor to the pathogenesis of the relapsing fever spirochete B. hermsii.

Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood.

It has been demonstrated that Borrelia hermsii, a causative agent of relapsing fever, produces a factor H (FH) and FH-like protein 1 (FHL-1) binding protein. The binding protein has been designated FhbA. To determine if FH/FHL-1 binding is widespread among B. hermsii isolates, a diverse panel of strains was tested for the FH/FHL-1 binding phenotype and FhbA production. Most isolates (23/24) produced FhbA and bound FH/FHL-1. Potential variation in FhbA among isolates was analyzed by DNA sequence analyses. Two genetically distinct FhbA types, designated fhbA1 and fhbA2, were delineated, and type-specific PCR primers were generated to allow for rapid differentiation. Pulsed-field gel electrophoresis and hybridization analyses demonstrated that all isolates that possess the gene carry it on a 200-kb linear plasmid (lp200), whereas isolates that lack the gene lack lp200 and instead carry an lp170. To determine if FhbA is antigenic during infection and to assess the specificity of the response, recombinant FhbA1 (rFhbA1) and rFhbA2 were screened with serum from infected mice and humans. FhbA was found to be expressed and antigenic and to elicit a potentially type-specific FhbA response. To localize the epitopes of FhbA1 and FhbA2, truncations were generated and screened with infection serum. The epitopes were determined to be conformationally defined. Collectively, these analyses indicate that FH/FHL-1 binding is a widespread virulence mechanism for B. hermsii and provide insight into the genetic and antigenic structure of FhbA. The data also have potential implications for understanding the epidemiology of relapsing fever in North America and can be applied to the future development of species-specific diagnostic tools.

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative complement pathway. The ability of FH-binding protein A (FhbA) to bind FH-like protein 1 (FHL-1) has not been assessed previously. In this study, using a whole-cell absorption assay, we demonstrated that B. hermsii absorbs both FH and FHL-1 from human serum. Consistent with this, affinity ligand binding immunoblot analyses revealed that FH constructs spanning short consensus repeats 1 to 7 and 16 to 20 bind to FhbA. To investigate the molecular basis of the interaction of FhbA with FH/FHL-1, recombinant FhbA truncated proteins were generated and tested for FH/FHL-1 binding. Binding required determinants located in both the N- and C-terminal domains of FhbA, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHL-1-binding pocket. To identify specific FhbA residues involved in binding, random mutagenesis was performed. These analyses identified a loop region of FhbA that may serve as a contact point for FH/FHL-1. The data presented here expand our understanding of the pathogenic mechanisms of the relapsing fever spirochetes and of the molecular nature of the interaction between FH/FHL-1 and FhbA.

Borrelia hermsii is the primary cause of tick-borne relapsing fever in North America. When its tick vector, Ornithodoros hermsi, acquires these spirochetes from the blood of an infected mammal, the bacteria switch their outer surface from one of many bloodstream variable major proteins (Vmps) to a unique protein, Vtp (Vsp33). Vtp may be critical for successful tick transmission of B. hermsii; however, the gene encoding this protein has been described previously in only one isolate. Here we identified and sequenced the vtp gene in 31 isolates of B. hermsii collected over 40 years from localities throughout much of its known geographic distribution. Seven major Vtp types were found. Little or no sequence variation existed within types, but between them significant variation was observed, similar to the pattern of diversity described for the outer surface protein C (OspC) gene in Lyme disease spirochetes. The pattern of sequence relatedness among the Vtp types was incongruent in two branches compared to two genomic groups identified among the isolates by multilocus sequence typing of the 16S rRNA, flaB, gyrB, and glpQ genes. Therefore, both horizontal transfer and recombination within and between the two genomic groups were responsible for some of the variation observed in the vtp gene. O. hermsi ticks were capable of transmitting spirochetes in the newly identified genomic group. Therefore, given the longevity of the tick vector and persistent infection of spirochetes in ticks, these arthropods rather than mammals may be the likely host where the exchange of spirochetal DNA occurs.

Unlike Borrelia burgdorferi, the relapsing fever agent Borrelia hermsii and the related Borrelia miyamotoi had purA and purB genes of the purine salvage pathway. These were located among the rRNA genes. Phylogenetic analysis indicated that these genes had a different evolutionary history than those of orthologs in other spirochetes.

Genome sequencing of the relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae identified three open reading frames (ORFs) on the chromosomes that contained internal, tandemly repeated amino acid sequences that were absent in the Lyme disease spirochete Borrelia burgdorferi. The predicted amino acid sequences of these genes (BH0209, BH0512, and BH0553) have hydrophobic N termini, indicating that these proteins may be secreted. B. hermsii transcribed the three ORFs in vitro, and the BH0512- and BH0553-encoded proteins (PBH-512 and PBH-553) were produced in vitro and in experimentally infected mice. PBH-512 and PBH-553 were on the spirochete's outer surface, and antiserum to these proteins reduced the adherence of B. hermsii to red blood cells. PCR analyses of 28 isolates of B. hermsii and 8 isolates of B. turicatae demonstrated polymorphism in each gene correlated with the number of repeats. Serum samples from relapsing fever patients reacted with recombinant PBH-512 and PBH-553, suggesting that these proteins are produced during human infection. These polymorphic proteins may be involved in the pathogenicity of these relapsing fever spirochetes and provide a mechanism for antigenic heterogeneity within their populations.

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.

A virulent, low-passage culture of a tick-derived strain of Borrelia garinii was subjected to serial in vitro passages, from which inoculations were made into C3H/HeN mice. A full display of pathogenicity was observed through passage 4, as measured by cultures of ear punch biopsy samples and internal organs and determination of tibiotarsal joint swelling. Decreased dissemination through skin and infection of internal organs were observed beginning at passage 6. These losses correlated with both the selection of clones harboring 21% less flagella than the parent strain, as seen by electron microscopy, and loss of the motility of the higher passages, as demonstrated by a swarm assay. However, during the chronic phase (3 months after infection), spirochetes were cultured from the bladder and kidney of a mouse inoculated with passage 12. The kidney isolate had the same number of flagella and motility as the original low-passage isolate. Although we can't exclude the possibility that other subtle variations may be arising given the uncloned nature of the isolate, we have found a strong association between loss of flagella and decreased invasiveness. Arthritogenicity progressively decreased with passages, so that only 12.5% of chronically infected mice inoculated with passage 29 still presented with joint swelling, concurrent with a decrease in the staining intensity in a Southern blot with a vlsE-based probe. These results suggest a multifactorial model in which the number of flagella drives the invasiveness of this agent, while plasmid-associated factors are responsible for triggering arthritogenicity.

Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation of serotype-specifying membrane proteins during mammalian infections. When B. hermsii is cultivated in broth medium, one serotype, 33, eventually predominates in the population. Serotype 33 has also been found to be dominant in ticks but not in mammalian hosts. We investigated the biology and genetics of two independently derived clonal populations of serotype 33 of B. hermsii. Both isolates infected immunodeficient mice, but serotype 33 cells were limited in number and were only transiently present in the blood. Probes for vsp33, which encodes the serotype-specifying Vsp33 outer membrane protein, revealed that the gene was located on a 53-kb linear plasmid and that there was only one locus for the gene in serotype 33. The vsp33 probe and probes for other variable membrane protein genes showed that expression of Vsp33 was determined at the level of transcription and that when the vsp33 expression site was active, an expression site for other variable proteins was silent. The study confirmed that serotype 33 is distinct from other serotypes of B. hermsii in its biology and demonstrated that B. hermsii can change its major surface protein through switching between two expression sites.

Borrelia hermsii, an agent of tick-borne relapsing fever, was found to contain multiple circular plasmids approximately 30 kb in size. Sequencing of a DNA library constructed from circular plasmid fragments enabled assembly of a composite DNA sequence that is homologous to the cp32 plasmid family of the Lyme disease spirochete, B. burgdorferi. Analysis of another relapsing fever bacterium, B. parkeri, indicated that it contains linear homologs of the B. hermsii and B. burgdorferi cp32 plasmids. The B. hermsii cp32 plasmids encode homologs of the B. burgdorferi Mlp and Bdr antigenic proteins and BlyA/BlyB putative hemolysins, but homologs of B. burgdorferi erp genes were absent. Immunoblot analyses demonstrated that relapsing fever patients produced antibodies to Mlp proteins, indicating that those proteins are synthesized by the spirochetes during human infection. Conservation of cp32-encoded genes in different Borrelia species suggests that their protein products serve functions essential to both relapsing fever and Lyme disease spirochetes. Relapsing fever borreliae replicate to high levels in the blood of infected animals, permitting direct detection and possible functional studies of Mlp, Bdr, BlyA/BlyB, and other cp32-encoded proteins in vivo.

Here, we describe the molecular and immunological characterization of the bdr gene family of Borrelia turicatae, a relapsing-fever spirochete. Nine bdr alleles belonging to two different subfamilies were sequenced and localized to linear plasmids. Anti-Bdr antiserum was generated and used to analyze Bdr expression in pre- and postinfection isogenic populations. The analyses presented here provide a detailed characterization of the Bdr proteins in a relapsing-fever spirochete species, enhancing our understanding of these proteins at the genus-wide level.

An outer surface lipoprotein of 22 kDa was identified in the avian pathogen Borrelia anserina Ni-NL by using antibody preparations reactive with bacterial surface-exposed proteins. Amino acid sequence analysis of the 22-kDa protein demonstrated 90% identity with VmpA of B. turicatae, suggesting that the protein belongs to the family of 20-kDa outer surface proteins of the genus Borrelia. All of the 60 chicks intramuscularly treated with antibodies specifically reacting with the 22-kDa protein and infected with strain Ni-NL were completely protected from infection, since no spirochetemia was detected, and from death. Control chicks were treated with immune sera raised against apathogenic strain B. anserina Es, which expresses a prominent 20-kDa polypeptide that is also a member of the Vmp family but does not cross-react immunologically with the 22-kDa protein of the Ni-NL strain. These animals, infected with B. anserina Ni-NL, showed a high degree of spirochetemia 10 days after infection, and all died between 14 and 21 days after infection. The results showed that the 22-kDa surface protein of B. anserina Ni-NL is a determinant of the pathogenic potential of the strain and also confirmed that only strain-specific antibodies are protective against B. anserina infection.

The tick-borne relapsing fever spirochete Borrelia hermsii evades the mammalian immune system by periodically switching expression among members of two multigene families that encode immunogenic, antigenically distinct outer surface proteins. The type strain, B. hermsii HS1, has at least 40 complete genes and pseudogenes that participate in this multiphasic antigenic variation. Originally termed vmp (for variable major protein) genes, they have been reclassified as vsp (for variable small protein) and vlp (for variable large protein) genes, based on size and amino acid sequence similarities. To date, antigenic variation in B. hermsii has been studied only in the type strain, HS1. Nucleotide sequence comparisons of 23 B. hermsii HS1 genes revealed five distinct groups, the vsp gene family and four subfamilies of vlp genes. We used PCR with family- and subfamily-specific primers, followed by restriction fragment length polymorphism analysis, to compare the vsp and vlp repertoires of HS1 and seven other B. hermsii isolates from Washington, Idaho, and California. This analysis, together with pulsed-field gel electrophoresis genome profiles, revealed that the eight isolates formed three distinct groups, which likely represent clonal lineages. Members of the three groups coexisted in the same geographic area, but they could also be isolated across large geographical distances. This population structure may result from immune selection by the host, as has been proposed for other pathogens with polymorphic antigens.

A polyreactive monoclonal antibody recognized a 38.5-kDa polypeptide with amino-terminal sequence identity to conserved regions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Borrelia burgdorferi, the Lyme disease agent, and Borrelia hermsii, an agent of American relapsing fever. This monoclonal antibody also recognized GAPDH from other pathogenic spirochetes and other prokaryotes and eukaryotes as well. GAPDH activity was detected in sonicates of both B. burgdorferi and B. hermsii but not in live, intact organisms, indicating the possibility of a subsurface localization for the Borrelia GAPDH activity. Degenerate primers constructed from highly conserved regions of gapdh of other prokaryotes successfully amplified this gene homolog in both B. burgdorferi and B. hermsii. Nuclei acid and deduced amino acid sequence analysis of the 838-bp probes for each borrelia indicated 93.9% identity between B. burgdorferi and B. hermsii at the amino acid level. Amino acid identities of B. burgdorferi and B. hermsii with Bacillus stearothermophilus were 59.2% and 58.8% respectively. Southern hybridization studies indicated that the gene encoding GAPDH is located on the chromosome of each borrella. In other bacterial species, GAPDH has other functions in addition to its traditional enzymatic role in the glycolytic pathway. GAPDH may play a similar role in borrelias.

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.

A comparison of the osp operon in 24 Lyme disease isolates, including representatives from each of the three established species, Borrelia burgdorferi, Borrelia garinii, and group VS461, was conducted. Several properties were assessed to determine whether the variability observed in this operon was reflective of the species of the isolate. At the transcriptional level, start site and Northern (RNA) blot analyses were conducted. B. garinii and VS461 group isolates were found to possess an untranslated leader sequence 6 nucleotides longer than that observed in B. burgdorferi isolates. By Northern blot analyses all Lyme disease isolates, except the B. garinii isolate VS102, were found to produce a polycistronic full-length ospAB message. Isolate VS102 produced a truncated message lacking the ospB portion of the transcript. Southern blot analyses suggest that the deletion occurred at the DNA level and was not due to a posttranscriptional event. Analysis of the outer surface proteins by two-dimensional gel electrophoresis demonstrated that the OspB isoelectric points were variable, with the OspB of B. garinii isolates exhibiting a pronounced acidic shift. The reactivity of different isolates to OspA and -B monoclonal antibodies and to a hyperimmune anti-ospAB serum was also variable. The results presented here demonstrate genotypic and phenotypic heterogeneity in the osp operon at both the inter- and intraspecies levels. The results have implications concerning the use of the osp genes or their gene products in the development of a Lyme disease vaccine, as diagnostic markers of Lyme disease, and in subtyping of Lyme disease isolates.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.

Three recombinant plasmids containing DNA from Borrelia coriaceae, the putative agent of epizootic bovine abortion, expressed antigens in Escherichia coli that reacted with antibodies specific for B. coriaceae. Two of the recombinants each expressed a single high-molecular-weight antigen. The third recombinant expressed three smaller antigens. The DNA inserts were sized and mapped. Hybridization of the cloned inserts to pulsed-field electrophoresis samples of B. coriaceae whole-cell DNA revealed the origin of two of the inserts to be located in linear plasmids. One of these, expressing three antigens, was located in a 210-kilobase linear plasmid. A second recombinant expressed a single antigen but hybridized to at least three distinct linear plasmids. The third clone also expressed a single antigen but was demonstrated to be chromosomal in origin.

OspA and OspB are major outer membrane proteins and antigens of the Lyme disease spirochete, Borrelia burgdorferi. We examined the organization of ospA and ospB, the genes encoding these proteins. The location and direction of transcription of each osp gene was determined by subcloning, deletion analysis, and transposon Tn5 mutagenesis. Transposon Tn5 insertions within the ospA gene abrogated expression of ospB, suggesting that these genes are transcribed from a common promoter. Northern blot analysis of mRNA from B. burgdorferi with two DNA probes individually specific for ospA or ospB identified a 2.2-kilobase transcript that hybridized with each probe. These studies indicate that the two osp genes of B. burgdorferi constitute a single transcriptional unit.

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.

In vitro cultivation of Borrelia burgdorferi, the etiologic agent of Lyme spirochetosis, allows for the isolation and growth of this bacterium from infected tissues. However, continuous cultivation in modified Kelly medium causes a reduction in the number of detectable plasmids and the loss of infectivity in the white-footed mouse, Peromyscus leucopus. In an unpassaged culture of B. burgdorferi, nine plasmids were present, including seven linear plasmids ranging in size from 49 to 16 kilobases (kb) and two circular plasmids of 27 and 7.6 kb. The 7.6-kb circular and 22-kb linear plasmids were no longer detectable in spirochetes noninfective in white-footed mice, suggesting that a gene(s) encoding for factors responsible for infection may be present on one or more of these extrachromosomal elements. Furthermore, changes in spirochetal proteins and lipopolysaccharide-like material were observed also during early cultivation and may be related to loss of infectivity.

By crossed immunoelectrophoresis and Western blotting (immunoblotting), it was shown that Borrelia burgdorferi expresses the 60-kilodalton Common Antigen (CA) that is cross-reactive with an equivalent antigen in a wide range of remotely related bacteria. B. burgdorferi CA is strongly immunogenic. A B. burgdorferi genomic library was constructed by using a plasmid cloning system. Escherichia coli recombinants were screened for expression of immunodominant B. burgdorferi antigens. One of the recombinant clones expressed the 60-kilodalton CA of B. burgdorferi. The DNA region encoding B. burgdorferi CA was localized on a 2.3-kilobase fragment of the plasmid pKH1. CA may have pathogenetic implications in Lyme borreliosis, since the CA of mycobacteria recently has been shown to play a role in the etiology of experimental autoimmune arthritis. The extensive cross-reactivity of this antigen may account for the low diagnostic specificity of the currently used serological tests in Lyme borreliosis.