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Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: ALG receives funding for central testing from Abbott and research grant support from Merck and Gilead, unrelated to the work performed here.

Pathogens persist in immunocompetent mammalian hosts using various strategies, including evasion of immune effectors by antigenic variation. Among highly antigenically variant bacteria, gene conversion is used to generate novel expressed variants from otherwise silent donor sequences. Recombination using oligonucleotide segments from multiple donors is a combinatorial mechanism that tremendously expands the variant repertoire, allowing thousands of variants to be generated from a relatively small donor pool. Three bacterial pathogens, each encoded by a small genome (< 1.2 Mb), illustrate this variant generating capacity and its role in persistent infection. Borrelia burgdorferi VlsE diversity is encoded and expressed on a linear plasmid required for persistence and recent experiments have demonstrated that VlsE recombination is necessary for persistence in the immunocompetent host. In contrast, both Treponema pallidum TprK and Anaplasma marginale Msp2 expression sites and donors are chromosomally encoded. Both T. pallidum and A. marginale generate antigenic variants in vivo in individual hosts and studies at the population level reveal marked strain diversity in the variant repertoire that may underlie pathogen strain structure and the capacity for re-infection and heterologous strain superinfection. Here, we review gene conversion in bacterial antigenic variation and discuss the short- and long-term selective pressures that shape the variant repertoire.

Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies). We studied a group of six recombinant T. pallidum antigens for their sensitivities and specificities with sera from individuals with syphilis (n = 43), relapsing fever (n = 8), Lyme disease (n = 8), and leptospirosis (n = 9) and from uninfected individuals (n = 15). Three recombinant proteins, Tp0155, Tp0483, and Tp0751, demonstrated sensitivity values that ranged from 28 to 42%. In contrast, three other recombinant proteins exhibited the following sensitivity and specificity values: Tp0453, 100% sensitivity and 100% specificity; Tp92 (Tp0326), 98% sensitivity and 97% specificity; and Gpd (Tp0257), 91% sensitivity and 93% specificity. Tp0453, Tp92, and Gpd also were recognized by sera from individuals with early primary syphilis that were nonreactive with the antilipoidal Venereal Disease Research Laboratory test. The reactivities of syphilis patient sera with Tp0453, Tp92, and Gpd were proportional to the titers of these sera with the treponemal test MHA-TP (microhemagglutination assay for T. pallidum). Thus, the recombinant T. pallidum antigens Tp0453, Tp92, and Gpd show promise as diagnostic antigens in the enzyme-linked immunosorbent assay-based assay.

In this study we describe the development of the T-cell response to a panel of Treponema pallidum antigens over the course of syphilis infection in the rabbit and determine whether these antigens induce the expression of Th1 cytokines. It was determined that the membrane proteins TpN17 and TpN47, as well as the endoflagellar sheath protein TpN37, induce strong proliferation responses through most of syphilis infection; Tromp1 induced only weak proliferative responses. An unexpected drop in proliferative response to these antigens at day 90 of infection, followed by a dramatic increase in response at day 180, suggests that there may be a secondary dissemination of T. pallidum which induces a recall response. Crude epitope mapping of TpN17 and TpN37 showed that multiple epitopes may be present on both antigens, which is likely a contributing factor in the immunodominance of these antigens. The T-cell response to the TpN37 molecule shows acquisition of newly recognized epitopes during the course of infection. Sonicated T. pallidum was found to induce the expression of interleukin-2 (IL-2) and gamma interferon and not IL-10 mRNA, showing that the general T-cell response to T. pallidum antigens in syphilis infection is biased towards the Th1 phenotype. Of the antigens tested, TpN37 appears to contribute the most to the Th1 cytokine response and therefore may play a key role in the clearance of T. pallidum from lesions.

Infectious syphilis, caused by the spirochete bacterium Treponema pallidum subsp. pallidum, remains a public health concern worldwide. The immune-response evasion mechanisms employed by T. pallidum are poorly understood, and prior attempts to identify immunoprotective antigens for subsequent vaccine design have been unsuccessful. Previous investigations conducted in our laboratory identified the T. pallidum glycerophosphodiester phosphodiesterase as a potential immunoprotective antigen by using a differential immunologic expression library screen. In studies reported here, heterologous expression of the T. pallidum glycerophosphodiester phosphodiesterase in Escherichia coli yielded a full-length, enzymatically active protein. Characterization of the recombinant molecule showed it to be bifunctional, in that it exhibited specific binding to human immunoglobulin A (IgA), IgD, and IgG in addition to possessing enzymatic activity. IgG fractionation studies revealed specific binding of the recombinant enzyme to the Fc fragment of human IgG, a characteristic that may play a role in enabling the syphilis spirochete to evade the host immune response. In further investigations, immunization with the recombinant enzyme significantly protected rabbits from subsequent T. pallidum challenge, altering lesion development at the sites of challenge. In all cases, animals immunized with the recombinant molecule developed atypical pale, flat, slightly indurated, and nonulcerative reactions at the challenge sites that resolved before lesions appeared in the control animals. Although protection in the immunized rabbits was incomplete, as demonstrated by the presence of T. pallidum in the rabbit infectivity test, glycerophosphodiester phosphodiesterase nevertheless represents a significantly immunoprotective T. pallidum antigen and thus may be useful for inclusion in an antigen cocktail vaccine for syphilis.

The 15-kDa lipoprotein of Treponema pallidum is a major immunogen during natural syphilis infection in humans and experimental infection in other hosts. The humoral and cellular immune responses to this molecule appear late in infection as resistance to reinfection is developing. One therefore might hypothesize that this antigen is important for protective immunity. This possibility is explored by using both genetic and antigenic approaches. Limited or no cross-protection has been demonstrated between the T. pallidum subspecies and strains or between Treponema species. We therefore hypothesized that if the 15-kDa antigen was of major importance in protective immunity, it might be a likely site of antigenic diversity. To explore this possibility, the sequences of the open reading frames of the 15-kDa gene have been determined for Treponema pallidum subsp. pallidum (Nichols and Bal-3 strains), T. pallidum subsp. pertenue (Gauthier strain), T. pallidum subsp. endemicum (Bosnia strain), Treponema paraluiscuniculi (Cuniculi A, H, and K strains), and a little-characterized simian isolate of Treponema sp. (Fribourg-Blanc strain). No significant differences in DNA sequences of the genes for the coding region of the 15-kDa antigen were found among the different species and subspecies studied. In addition, all organisms showed expression of the 15-kDa antigen as determined by monoclonal antibody staining. The role of the 15-kDa antigen in protection against homologous infection with T. pallidum subsp. pallidum Nichols was examined in rabbits immunized with a purified recombinant 15-kDa fusion protein. No alteration in chancre development was observed in immunized, compared to unimmunized, rabbits, and the antisera induced by the immunization failed to enhance phagocytosis of T. pallidum subsp. pallidum by macrophages in vitro. These results do not support a major role for this antigen in protection against syphilis infection.

Classifications of 48 atypical clinical isolates of Mobiluncus spp. were determined on the basis of biochemical reactions, morphology, antigenic profiles, and DNA hybridization studies. Two new subgroups with unique antigenic profiles are described. Like typical Mobiluncus species, the antigenic variant of M. mulieris is associated with bacterial vaginosis. The atypical isolates of M. curtisii were frequently recovered from women with normal vaginal flora and were also recovered from sterile body sites. These isolates may be incorrectly identified if current biochemical and morphological criteria are used for identification. Gram stain morphology, however, correctly identifies these isolates to the species level. The characterization of these atypical isolates has important implications for future investigations in which serological methods are used for diagnosis, epidemiology, and determination of pathogenicity of Mobiluncus spp.

The protein and antigenic profiles of the American Type Culture Collection type strains of Mobiluncus species and those of 114 clinical isolates were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and immunoblotting with homologous polyvalent antisera. The majority of isolates (82%) possessed characteristic protein profiles and could be identified to the species level by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The major protein bands were also antigenic, and some antigenic cross-reactivity was noted between the two Mobiluncus species. All of the isolates were examined for reactivity with a panel of 12 monoclonal antibodies previously prepared against the type strains. While 56 of 60 clinical isolates of Mobiluncus curtisii (93%) reacted with one or more of the monoclonal antibodies, only 23 of 54 clinical isolates which were identified as Mobiluncus mulieris by biochemical methods (48%) reacted with one or more of the monoclonal antibodies. One of the 4 M. curtisii isolates (25%) and 11 of the 31 M. mulieris isolates (35%) which did not react with the monoclonal antibodies also had atypical protein profiles. These results demonstrate a high degree of heterogeneity in the protein and antigenic profiles of Mobiluncus isolates and suggest that further taxonomic division may be appropriate.

Protein antigens of Treponema pallidum, Nichols strain, and Treponema pertenue, Gauthier strain, were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Treponemal proteins were solubilized in 1% sodium dodecyl sulfate, electrophoresed on 12.5% polyacrylamide gels, and either stained with Coomassie brilliant blue or electrophoretically transferred to nitrocellulose paper. These antigen blots were incubated with sera from rabbits infected with either T. pallidum or T. pertenue and 125I-labeled staphylococcal protein A and exposed to X-ray film for visualization of antigenic molecules. Protein profiles of each organism separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue showed no distinguishable differences. Antigenic profiles as determined by Western blots were similar with two exceptions. A 39,500-dalton band was present on T. pertenue but absent from T. pallidum, and a 19,000-dalton band was present on T. pallidum but absent from T. pertenue (although two additional antigenic bands at 21,000 and 18,000 daltons were seen on T. pertenue). Because these differences were detected by using antisera raised against either T. pallidum or T. pertenue, these molecules must contain some antigenic determinants in common despite their differences in molecular weight.

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

The antigenic cross-reactivity between Treponema pallidum and several pathogenic members of the family Spirochaetaceae was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Blots of T. pallidum antigens were incubated with antiserum from rabbits infected or immunized with T. pallidum, Treponema paraluiscuniculi, Treponema hyodysenteriae (strains B204 and T22), Borrelia hermsii serotype 7, or Leptopsira interrogans serogroup Canicola. T. pallidum contained 22 antigenic molecules ranging from 85,000 to 12,000 daltons which were recognized by serum from rabbits infected with T. pallidum. Serum from rabbits infected with T. paraluiscuniculi cross-reacted with 21 of these molecules and faintly reacted with a band at 15,000 daltons which was not recognized by anti-T. pallidum serum. Antisera directed against strains B204 and T22 of T. hyodysenteriae cross-reacted with 11 and 10 antigens of T. pallidum, respectively. B. hermsii and L. interrogans serogroup Canicola antisera detected 11 and 10 treponemal antigens, respectively. Many of the T. pallidum antigens detected by antisera against T. hyodysenteriae, B. hermsii, or L. interrogans serogroup Canicola have been previously identified as containing moieties also found on the nonpathogenic Treponema phagedenis, biotype Reiter, and may therefore represent group antigens common to members of the family Spirochaetaceae.

The contribution of individual specific molecules of Treponema pallidum subspecies pallidum to cellular immunity in experimental syphilis was evaluated by combining the techniques of Ag identification and purification with the lymphocyte proliferation assay. Proliferative responses of splenic lymphocytes from syphilitic rabbits to complex treponemal Ag and Con A were vigorous throughout the course of intratesticular infection (6, 10, 17, 30, and 210 days). Normal rabbits did not respond to any treponemal preparations and all rabbits failed to respond to normal rabbit testicular Ag (NRT). Seven defined treponemal Ag (47 kDa, 37 kDa, 35, 33-kDa, 30-kDa, 14 kDa, and 12 kDa) stimulated lymphocytes from infected rabbits. Cellular responses to the 37-kDa and 30-kDa fractions were evident by day 6 of infection and responses to the 35, 33-kDa and 14-kDa Ag were first detected on day 10; responsiveness to these Ag continued throughout the observation period. Cellular responses to the 47-kDa molecule were detectable but lower when compared with other individual Ag. Responsiveness to the 12-kDa Ag was not evident until 7 mo postinfection. Specific immunoblot reactivity of serum from rabbits used in this study generally correlated with the development of cellular reactivity to individual Ag of T. pallidum.

The major antigens of Treponema pallidum, Nichols strain were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting techniques. Organisms were extracted from rabbit testes, purified by differential centrifugation, washed, and disrupted by sonication before solubilization in SDS buffer, SDS-PAGE analysis on 12.5% gels revealed at least 35 distinguishable polypeptides with molecular weights between 14,000 and 100,000. Proteins were electrophoretically transferred from gels to nitrocellulose paper and were stained with pooled rabbit anti-T. pallidum serum (IRS) and 125I-protein A. At least eight distinct antigenic molecules were revealed, with the major component residing at 48,000 daltons. Extensive washing of T. pallidum before solubilization did not alter the antigenic profile. Five molecules with common treponemal antigenic determinants were identified both by staining blots of T. phagedenis, biotype Reiter with IRS and by staining blots of T. pallidum with rabbit anti-Reiter antiserum. Three putative T. pallidum-specific antigens were identified by staining T. pallidum with IRS that had been extensively adsorbed with Reiter treponemes.

Thirteen hybrid cell lines which produce mouse monoclonal antibodies to Treponema pallidum, the causative agent of syphilis, have been established. All of the monoclonal antibodies react with T. pallidum, Nichols strain, in ELISA and in immunofluorescence assays, but do not react with normal rabbit testicular tissue in the ELISA. Two of these antibodies were demonstrated to react with the nonpathogenic treponemes T. phagedenis, biotype Reiter, T. refringens (Noguchi strain), T. vincentii, and T. denticola (strains 11 and W), as well as with Borrelia recurrentis, Leptospira interrogans, serogroup Canicola, and the swine pathogen T. hyodysenteriae. The remaining 11 antibodies react with four recently isolated strains of T. pallidum, but with none of the related nonpathogens nor with Borrelia or Leptospira. Thus, our results to date indicate that these monoclonal antibodies may identify antigenic determinants that are specific either for T. pallidum alone or for those treponemes which are pathogenic for humans. The molecular specificities of six of the 13 antibodies were determined by Western blotting. We anticipate potential usefulness of these antibodies in the investigation of the antigenic structure of T. pallidum, the taxonomic study of the pathogenic and nonpathogenic treponemes, and in the diagnosis of syphilis.

IgG and IgM antibody specificities for antigens of Treponema pallidum Nichols strain were determined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the western blot technique in sera from patients with untreated syphilis, normal persons, persons with biologic false-positive tests for syphilis, and sexual contacts of persons with infectious syphilis. IgG reactivities of sera from individuals with primary syphilis varied considerably but consistently exhibited strong reactivity to a 48-kilodalton band. Sera from patients with secondary and early latent syphilis uniformly demonstrated reactivity to 22 separate polypeptide antigens; decreased reactivity was seen in late latent syphilis. Normal and biologic false-positive sera showed weak IgG reactivity against none to 12 polypeptides. Sera from asymptomatic contacts of persons with infectious syphilis showed reactivity to a varying number of treponemal antigens, including some reactions not seen with normal sera. IgM reactivity was most prominent in secondary syphilis but was demonstrable at all stages of disease.