Condon, and C. is thought not to be infectious for humans (14). In contrast, the three subspecies of naturally infect humans but can also experimentally infect rabbits and other mammals. naturally infects humans but is unable to multiply in rabbits or other nonprimates (28). is the etiologic agent of rabbit venereal syphilis. It was identified in 1913, only 8 years after subsp. (hereafter), the etiologic agent of human venereal syphilis, was discovered (3, 25). By dark-field microscopy, immunoassay, and electron microscopy, and the other pathogenic treponemes are morphologically indistinguishable (17) and appear to induce similar immune responses and histopathologic changes. Furthermore, and antisera for antigens (2, 26). Despite this apparently high level of relatedness, the pathogenic treponemes have different host specificities and cause clinically distinct diseases. Infections with all these treponemal species are chronic, and while homologous immunity exists, there is no cross-protection between species of pathogenic treponemes and only partial cross-protection within species. These facts indicate that there are small but important differences between the virulence factors and antigens expressed by the different pathogenic treponemes. Paralogous gene families arise during evolution by gene duplication and may yield new gene functions and new expression patterns (20-22). Initial analysis of the genome sequence of Nichols strain revealed 48 potential virulence genes, among them a novel family of 12 paralogous genes (genes, for repeat) (13). The genes account for 2% of the small genome. The family is divided into three subfamilies by their predicted amino acid homology: subfamily I (TprC, TprD, TprF, and TprI), subfamily II (TprE, TprG, and TprJ), and subfamily III (TprA, TprB, TprH, TprK, and TprL). The subfamily I and subfamily II proteins have conserved NH2- and COOH-terminal regions, whereas the central domains vary sequence and length. Within subfamily I, TprC and TprD are identical in the Nichols strain, and TprF lacks dmDNA31 a central variable domain and the conserved COOH-terminal region due to a frameshift. Subfamily III members are comparatively less homologous to each other and to the other Tpr proteins. dmDNA31 It dmDNA31 has been shown that TprK, TprI, and TprF are the targets of a strong humoral and cellular immune response during syphilis infection in the rabbit model, and immunization with recombinant peptides significantly alters lesion development (4, 23, 27). In addition, TprK contains a cleavable leader sequence, possesses multiple alleles in isolates, and undergoes antigenic variation during syphilis infection (6, 7, 16). This suggests an important role for the Tpr antigens during syphilis infection. Among the several approaches used to identify virulence factors are intra- and interspecies comparisons to determine which antigens are conserved and which are variable among different strains or species (30). Weinstock et al. reported that, while genes are highly conserved among human syphilis strains, most of the differences between the subsp. and subsp. subspecies involved the genes (30). Despite the high level of relatedness and the natural ability to infect rabbits, is considered not to be infectious for humans (14). The host specificity shown by this microorganism in association with the similarities to syphilitic infection make a unique comparison species for understanding which genetic determinants contribute dmDNA31 to pathogenesis or virulence in human syphilis. For this reason, we studied the gene family in the rabbit treponeme to determine the sequence architecture of and immune responses to these antigens. In this study, we characterized the gene family of propagation and DNA extraction. Cuniculi A strain was provided by Paul Hardy and Ellen Nell (Johns Hopkins University, Baltimore, Md.), propagated intratesticularly in New Zealand White rabbits and harvested as described elsewhere (19). Before infection, each rabbit had been serologically tested to rule out a naturally occurring infection with for 10 min at room temperature); the dmDNA31 CYFIP1 supernatants were spun in a microcentrifuge for 30 min at 12,000 at 4C. The pellet was resuspended in 200 l of 1 1 lysis buffer (10 mM Tris [pH 8.0] 0.1 M EDTA, 0.5% sodium dodecyl sulfate). DNA extraction was performed as previously described (5) with the QIAamp DNA minikit (Qiagen Inc., Chatsworth, Calif.), taking standard precautions to prevent cross-contamination between samples. PCR amplification, cloning, sequencing, and sequence analysis. The genome sequence (13) was used to design primers in the 5- and 3-flanking regions of the Nichols genes to amplify the related DNA areas. The primers utilized for amplification are outlined in Table ?Table1.1. Each PCR.