Comparative analysis of antibody responses to outer surface protein (Osp)A and OspC in dogs vaccinated with Lyme disease vaccines.

Lyme disease (LD), the most common tick-borne disease of canines and humans in N. America, is caused by the spirochete Borreliella burgdorferi. Subunit and bacterin vaccines are available for the prevention of LD in dogs. LD bacterin vaccines, which are comprised of cell lysates of two strains of B. burgdorferi, contain over 1000 different proteins and cellular constituents. In contrast, subunit vaccines are defined in composition and consist of either outer surface protein (Osp)A or OspA and an OspC chimeritope. In this study, we comparatively assessed antibody responses to OspA and OspC induced by vaccination with all canine bacterin and subunit LD vaccines that are commercially available in North America. Dogs were administered a two-dose series of the vaccine to which they were assigned (3 weeks apart): Subunit-AC, Subunit-A, Bacterin-1, and Bacterin-2. Antibody titers to OspA and OspC were determined by ELISA and the ability of each vaccine to elicit antibodies that recognize diverse OspC proteins (referred to as OspC types) assessed by immunoblot. While all of the vaccines elicited similar OspA antibody responses, only Subunit-AC triggered a robust and broadly cross-reactive antibody response to divergent OspC proteins. The data presented within provide new information regarding vaccination-induced antibody responses to key tick and mammalian phase antigens by both subunit and bacterin LD canine vaccine formulations.

The Leptospiral General Secretory Protein D (GspD), a secretin, elicits complement-independent bactericidal antibody against diverse Leptospira species and serovars.

Leptospirosis, the most common zoonotic infection worldwide, is a multi-system disorder affecting the kidney, liver, and lungs. Infections can be asymptomatic, self-limiting or progress to multi-organ system failure and pulmonary hemorrhage. The incidence of canine and human leptospirosis is steadily increasing worldwide. At least sixty-four Leptospira species and several hundred lipopolysaccharide-based serovars have been defined. Preventive vaccines are available for use in veterinary medicine and limited use in humans in some countries. All commercially available vaccines are bacterin formulations that consist of a combination of laboratory cultivated strains of different lipopolysaccharide serotypes. The development of a broadly protective subunit vaccine would represent a significant step forward in efforts to combat leptospirosis in humans, livestock, and companion animals worldwide. Here we investigate the potential of General secretory protein D (GspD; LIC11570), a secretin, to serve as a possible antigen in a multi-valent vaccine formulation. GspD is conserved, expressed in vitro, antigenic during infection and elicits antibody with complement independent bactericidal activity. Importantly, antibody to GspD is bactericidal against diverse Leptospira species of the P1 subclade. Epitope mapping localized the bactericidal epitopes to the N-terminal N0 domain of GspD. The data within support further exploration of GspD as a candidate for inclusion in a next generation multi-protein subunit vaccine.

Antibody profiling of a Borreliella burgdorferi (Lyme disease) C6 antibody positive, symptomatic Rottweiler and her pups.

Lyme disease (LD) is a tick-transmitted disease caused by Borreliella burgdorferi (Bb). Temporal studies of maternal antibody (Ab) profiles in Bb infected pregnant dogs and their pups have not been conducted. In this study, Ab profiles of a client-owned Bb C6 Ab positive Rottweiler and her nine pups were assessed. The dam presented with lameness 12 days prior to parturition and was C6 Ab positive with a Quant C6 Ab concentration of 237U/mL. Treatment with amoxicillin was initiated and 11 days later nine pups were delivered. Screening of the sera from the dam and pups against Bb cell lysates and a panel of antigens revealed similar immunoreactivity profiles. While antigen-specific IgG and IgM reactivity persisted in the dam for at least 7 months, a rapid decline in IgG specific for BBA36, BBK53, BB0238, BBA73 and outer surface protein (Osp) E in the pups occurred between days 29 and 52 post-parturition. In contrast, Ab specific for DbpA and the diagnostic antigens VlsE (C6) and OspF, remained elevated in the pups. Sera from the dam displayed potent complement-dependent bactericidal activity against Bb. Sera from the pups was also bactericidal but primarily through a complement-independent mechanism. Lastly, single dose vaccination of the dam at day 51 post-parturition with a LD subunit vaccine consisting of OspA and an OspC chimeritope triggered a broad anti-OspC Ab response indicative of an anamnestic response. Although this study focused on a single case, these findings add to our knowledge of maternal Ab profiles and will aid the interpretation of serological assays in pups delivered by a Bb C6 Ab positive dog.

Plasminogen binding and degradation by Treponema denticola: Identification of the plasminogen binding interface on the FhbB protein.

Treponema denticola is a proteolytic-anaerobic spirochete whose abundance in the subgingival crevice correlates with periodontal disease severity. Treponema denticola evades serum-mediated killing through the binding of factor H (FH), a negative regulator of the complement system. The T. denticolaFH receptor has been identified as FhbB, an 11.4kDa immunodominant lipoprotein. Three distinct subfamilies of FhbB proteins have been delineated and designated as FhbB1, FhbB2 and FhbB3. In this study we demonstrate that all FhbB variants bind human plasminogen (Plg). Competitive binding analyses revealed that FH and Plg do not compete for binding. Binding studies with FhbB135405 site-directed amino acid substitution mutants demonstrated that the interaction domains for FH and Plg on FhbB are separable. Inhibition of Plg-FhbB binding by ε-aminocaproic acid (a lysine analog) indicates that binding is mediated by electrostatic interactions that presumably occur with Lys binding sites contained within Plg "Kringle" domains 1, 2, 4 or 5. Similar to that demonstrated for FH, Plg can also serve as a substrate for the T. denticola protease, dentilisin. The in vivo consequences of dentilisin-mediated cleavage of Plg remained to be determined. The data presented demonstrate that FhbB is a multi-functional protein that may contribute to virulence through several mechanisms including immune evasion, manipulation of the host immune response, adherence or tissue invasion.

The Treponema denticola AtcR LytTR domain-containing response regulator interacts with three architecturally distinct promoter elements: implications for understanding the molecular signaling mechanisms that drive the progression of periodontal disease.

Treponema denticola is an oral spirochete and periopathogen that transitions from low abundance in healthy subgingival crevices to high abundance in periodontal pockets. The T. denticola response regulator AtcR harbors the relatively rare, LytTR DNA-binding domain. LytTR domain containing response regulators control critical transcriptional responses required for environmental adaptation. Using a multi-step bioinformatics approach, 26 strong lytTR recognition motifs were identified in the genome of T. denticola strain 35405. Electrophoretic mobility shift assays demonstrated that AtcR binds to these recognition motifs. High specificity-high affinity complexes formed with phosphorylated AtcR. The LytTR recognition sequences were found to exist in three distinct promoter architectures designated as LytTR1, LytTR2 and LytTR3 promoters. LytTR1 and LytTR2 promoters harbor σ(54) binding sites. The functional diversity of the proteins encoded by the putative AtcR regulon suggests that AtcR sits at the top of a regulatory cascade that plays a central role in facilitating T. denticola's ability to adapt to changing environmental conditions and thrive in periodontal pockets.

Analysis of the complement sensitivity of oral treponemes and the potential influence of FH binding, FH cleavage and dentilisin activity on the pathogenesis of periodontal disease.

Treponema denticola, a periopathogen, evades complement-mediated killing by binding the negative complement regulatory protein factor H (FH) to its surface via the FhbB protein. Paradoxically, bound FH is cleaved by T. denticola's dentilisin protease, a process hypothesized to trigger localized dysregulation of complement activation in periodontal pockets. The ability of other oral treponemes to evade complement-mediated killing and bind and cleave FH has not been assessed. In this report, we demonstrate that representative isolates of Treponema socranskii, Treponema medium, Treponema pectinovorum and Treponema maltophilum are also serum resistant, whereas Treponema vincentii and Treponema amylovorum are serum sensitive. Although T. denticola's ability to evade complement-mediated killing is strictly dependent on FH binding, other serum-resistant treponemal species lack FhbB and do not bind FH, indicating an FH-independent mechanism of complement evasion. To assess the influence of FhbB sequence variation on FH binding and cleavage by T. denticola, fhbB sequences were determined for 30 isolates. Three distinct phyletic types were identified. All T. denticola strains bound FH and were serum resistant, but differences in binding kinetics, dentilisin activity and FH cleavage ability were observed. Based on these analyses, we hypothesize that the composition of the T. denticola population is a determining factor that influences the progression and severity of periodontal disease.

Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: implications for Lyme disease vaccine and diagnostic assay design.

In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n=2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.

Sequence divergence in the Treponema denticola FhbB protein and its impact on factor H binding.

Treponema denticola is an anaerobic spirochete whose abundance in the subgingival crevice correlates with the development and severity of periodontal disease. The ability of T. denticola to survive and thrive in the hostile environment of the periodontal pocket is due, at least in part, to its ability to bind factor H (FH), a negative regulator of the alternative complement pathway. The FH binding protein of T. denticola has been identified as FhbB and its atomic structure has been determined. The interaction of FH with T. denticola is unique in that FH bound to the cell surface is cleaved by the T. denticola protease, dentilisin. It has been postulated that FH cleavage by T. denticola leads to immune dysregulation in periodontal pockets. In this study, we conduct a comparative assessment of the sequence, properties, structure and ligand binding kinetics of the FhbB proteins of strains 33521 and 35405. The biological outcome of the interaction of these strains with FH could differ significantly as 33521 lacks dentilisin activity. The data presented here offer insight into our understanding of the interactions of T. denticola with the host and its potential to influence disease progression.

Identification of the primary mechanism of complement evasion by the periodontal pathogen, Treponema denticola.

Treponema denticola, a periodontal pathogen, binds the complement regulatory protein Factor H (FH). Factor H binding protein B (FhbB) is the sole FH binding protein produced by T. denticola. The interaction of FhbB with FH is unique in that FH is bound to the cell and then cleaved by the T. denticola protease, dentilisin. A ∼ 50-kDa product generated by dentilisin cleavage is retained at the cell surface. Until this study, a direct role for the FhbB-FH interaction in complement evasion and serum sensitivity had not been demonstrated. Here we assess the serum resistance of T. denticola strain 35405 (Td35405wt) and isogenic mutants deficient in dentilisin (Td35405-CCE) and FhbB production (Td35405ΔfhbB), respectively. Both dentilisin and FhbB have been postulated to be key virulence factors that mediate complement evasion. Consistent with conditions in the subgingival crevice, an environment with a significant concentration of complement, Td35405wt was resistant to serum concentrations as high as 25%. Deletion of fhbB (Td35405ΔfhbB), which resulted in the complete loss of FH binding ability, but not inactivation of dentilisin activity (Td35405-CCE), rendered T. denticola highly sensitive to 25% human serum with 80% of the cells being disrupted after 4 h of incubation. Heat treatment of the serum to inactivate complement confirmed that killing was mediated by complement. These results indicate that the FH-FhbB interaction is required for serum resistance whereas dentilisin is not. This report provides new insight into the novel complement evasion mechanisms of T. denticola.

Molecular signaling mechanisms of the periopathogen, Treponema denticola.

In the healthy subgingiva, oral treponemes account for a small percentage of the total bacteria. However, in diseased periodontal pockets, treponemes thrive and become a dominant component of the bacterial population. Oral treponemes are uniquely adept at capitalizing on the environmental conditions that develop with periodontal disease. The molecular basis of adaptive responses of oral treponemes is just beginning to be investigated and defined. The completion of several treponeme genome sequences and the characterization of global regulatory systems provide an important starting point in the analysis of signaling and adaptive responses. In this review, we discuss existing literature focused on the genetic regulatory mechanisms of Treponema denticola and present an overview of the possible roles of regulatory proteins identified through genome analyses. This information provides insight into the possible molecular mechanisms utilized by oral spirochetes to survive in the periodontal pocket and transition from a minor to a dominant organism.

The Hpk2-Rrp2 two-component regulatory system of Treponema denticola: a potential regulator of environmental and adaptive responses.

Treponema denticola levels in the gingival crevice become elevated as periodontal disease develops. Oral treponemes may account for as much as 40% of the total bacterial population in the periodontal pocket. The stimuli that trigger enhanced growth of T. denticola, and the mechanisms associated with the transmission of these signals, remain to be defined. We hypothesize that the T. denticola open reading frames tde1970 (histidine kinase) and tde1969 (response regulator) constitute a functional two-component regulatory system that regulates, at least in part, responses to the changing environmental conditions associated with the development of periodontal disease. The results presented demonstrate that tde1970 and tde1969 are conserved, universal among T. denticola isolates and transcribed as part of a seven-gene operon in a growth-phase-dependent manner. tde1970 undergoes autophosphorylation and transfers phosphate to tde1969. Henceforth, the proteins encoded by these open reading frames are designated as Hpk2 and Rrp2 respectively. Hpk2 autophosphorylation kinetics were influenced by environmental conditions and by the presence or absence of a PAS domain. It can be concluded that Hpk2 and Rrp2 constitute a functional two-component system that contributes to environmental sensing.

Evidence that the BBA68 protein (BbCRASP-1) of the Lyme disease spirochetes does not contribute to factor H-mediated immune evasion in humans and other animals.

BBA68 (BbCRASP-1) of the Lyme disease spirochetes binds human factor H (FH) and FH-like protein 1 (FHL-1). Here we assess transcription of the BBA68 gene and production of BBA68 in infected mice and humans using real-time reverse transcriptase PCR and immunoblotting. The species specificity of FH binding to BBA68 was also tested. The data suggest that BBA68 does not play an important role in immune evasion in animals.

Evidence for the contribution of point mutations to vlsE variation and for apparent constraints on the net accumulation of sequence changes in vlsE during infection with Lyme disease spirochetes.

In the Lyme disease spirochetes, both the ospE and vlsE gene families have been demonstrated to undergo sequence variation during infection. To further investigate the mechanisms associated with the generation of vls variation, single-nucleotide polymorphism and subsequent DNA sequence analyses were performed on the vlsE gene and its paralog, BBJ51, a related gene with a frameshift mutation. These analyses focused on a series of postinfection clonal populations obtained from mice infected with Borrelia burgdorferi B31MIpc or its clonal derivative, B31MIc53. vlsE, but not BBJ51, was found to undergo sequence changes during infection. Consistent with that reported previously (J.-R. Zhang et al., Cell 89:275-285, 1997) many of the sequence changes appear to have arisen through gene conversion events and to be localized to the variable regions of vlsE. However, analysis of the vlsE nucleotide sequences revealed that some sequence changes were the result of point mutations, as these changes did not have potential contributing sources in the vls cassettes. To determine if sequence changes accumulate in vlsE over long-term infection, the vlsE genes of clonal populations recovered after 7 months of infection in mice were analyzed. While new sequence changes developed, a significant number of these changes resulted in the restoration of the vlsE sequence of the original infecting clone. In addition, we noted that some positions within the variable regions (VR) are stable even though the cassettes possess residues that could contribute to sequence variation through gene conversion. These analyses suggest that the total number of amino acid sequence changes that can be maintained by VlsE levels off during infection. In summary, in this report we demonstrate that the development of point mutations serves as a second mechanism by which vlsE sequence variation can be generated and that the capacity for vlsE variation, while still significant, is less than previously postulated.

Demonstration of the genetic stability and temporal expression of select members of the lyme disease spirochete OspF protein family during infection in mice.

Infection with Lyme disease spirochetes can be chronic. This suggests that the spirochetes are capable of immune evasion. In a previous study we demonstrated that the ospE gene family, which is one of three gene families whose members are flanked at their 5' end by the highly conserved upstream homology box (UHB) element, undergoes mutation and rearrangement during infection. This results in the generation of antigenically distinct variants that may contribute to immune evasion. In this study we have assessed the genetic stability of the UHB-flanked ospF gene family during infection in mice. Using postinfection clonal populations of Borrelia burgdorferi B31MI, PCR amplicons were generated for three members of the ospF gene family after a 3-month infection time frame. The amplicons were analyzed by single-nucleotide polymorphism pattern analysis and DNA sequencing. Members of the ospF gene family were found to be stable during infection, as no mutations or rearrangements were detected. An analysis of the humoral immune response to these proteins during infection revealed that the immune response to each is specific and that there is a delayed humoral immune response to some OspF protein family members. These analyses suggest that there is a temporal component to the expression of these genes during infection. In addition to a possible contribution to immune evasion, members of the OspF protein family may play specific roles at different stages of infection.

Analysis of mechanisms associated with loss of infectivity of clonal populations of Borrelia burgdorferi B31MI.

Numerous studies have provided suggestive evidence that the loss of plasmids correlates with the loss of infectivity of the Lyme disease spirochetes. In this study we have further investigated this correlation. Clonal populations were obtained from the skin of a mouse infected for 3 months with a clonal population of Borrelia burgdorferi B31MI. The complete plasmid compositions of these populations were determined using a combination of PCR and Southern hybridization. The infectivities of clones differing in plasmid composition were tested using the C3H-HeJ murine model for Lyme disease. While several clones were found to be noninfectious, a correlation between the loss of a specific plasmid and loss of infectivity in the clones analyzed in this report was not observed. While it is clear from recent studies that the loss of some specific plasmids results in attenuated virulence, this study demonstrates that additional mechanisms also contribute to the loss of infectivity.

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.

The bdr gene families of the Lyme disease and relapsing fever spirochetes: potential influence on biology, pathogenesis, and evolution.

Species of the genus Borrelia cause human and animal infections, including Lyme disease, relapsing fever, and epizootic bovine abortion. The borrelial genome is unique among bacterial genomes in that it is composed of a linear chromosome and a series of linear and circular plasmids. The plasmids exhibit significant genetic redundancy and carry 175 paralogous gene families, most of unknown function. Homologous alleles on different plasmids could influence the organization and evolution of the Borrelia genome by serving as foci for interplasmid homologous recombination. The plasmid-carried Borrelia direct repeat (bdr) gene family encodes polymorphic, acidic proteins with putative phosphorylation sites and transmembrane domains. These proteins may play regulatory roles in Borrelia. We describe recent progress in the characterization of the Borrelia bdr genes and discuss the possible influence of this gene family on the biology, pathogenesis, and evolution of the Borrelia genome.

Molecular and immunological analyses of the Borrelia turicatae Bdr protein family.

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.

Mutation and recombination in the upstream homology box-flanked ospE-related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection.

The ospE gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The ospE genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box, or UHB, element. Earlier analyses in our lab demonstrated that ospE-related genes are characterized by defined hypervariable domains (domains 1 and 2) that are predicted to be hydrophilic, surface exposed, and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to ospE diversity and thus ultimately to antigenic variation. Using an isogeneic clone of Borrelia burgdorferi B31G (designated B31Gc1), we demonstrate that the ospE-related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE-related proteins in immune system evasion.

Evolutionary and molecular analyses of the Borrelia bdr super gene family: delineation of distinct sub-families and demonstration of the genus wide conservation of putative functional domains, structural properties and repeat motifs.

B. turicatae, a causative agent of relapsing fever, carries a polymorphic gene family that is homologous to the bdr gene family of the Lyme disease spirochetes (previously referred to as the rep+ or ORF-E gene family). Here we demonstrate that bdr related genes are widely distributed among pathogenic Borrelia species and exist as large, polymorphic, plasmid carried, gene families. Twenty distinct bdr alleles were identified in isolates of the relapsing fever spirochete, B. hermsii, and were localized to linear plasmids. Cloning and sequence analyses demonstrate that the putative Bdr functional domains (i.e. the phosphorylation motifs and the transmembrane C-terminal domain) are conserved across the genus while other regions of these proteins exhibit variability. An assessment of the evolutionary relationships among all known Bdr protein sequences obtained from five pathogenic Borrelia species revealed that there are distinct Bdr sub-families. The recognition of distinct phyletic clusters serves as the basis of a revised and simplified nomenclature for the bdr proteins that can be applied genus wide. At the biological level the delineation of multiple bdr sub-families within isogeneic populations raises the possibility that there may be functional partitioning among alleles. In summary, the distribution and conservation of the Bdr proteins suggests that they are important in the biology/pathogenesis of the Borrelia at the genus wide level.