We investigated the evolution of 6 genes from the Treponema pallidum repeat (tpr) gene family, which encode potential virulence factors and are assumed to have evolved through gene duplication and gene conversion events. The 6 loci (tprC, D, G, J, I, and K) were sequenced and analyzed in several members of the genus Treponema, including the 3 subspecies of human T. pallidum (T. pallidum subsp. pallidum, pertenue, and endemicum), Treponema paraluiscuniculi (rabbit syphilis), and the unclassified Fribourg-Blanc (simian) isolate. Phylogenetic methods, recombination analysis, and measures of nucleotide diversity were used to investigate the evolutionary history of the tpr genes. Numerous instances of gene conversion were detected by all 3 methods including both homogenizing gene conversion that involved the entire length of the sequence as well as site-specific conversions that affected smaller regions. We determined the relative age and directionality of the gene conversion events whenever possible. Our data are also relevant to a discussion of the evolution of the treponemes themselves. Higher levels of variation exist between the human subspecies than within them, supporting the classification of the human treponemes into 3 subspecies. In contrast to published theories, the divergence and diversity of T. pallidum subsp. pertenue relative to the other subspecies does not support a much older origin of yaws at the emergence of modern human, nor is the level of divergence seen in T. pallidum subsp. pallidum consistent with a very recent (,500 years) origin of this subspecies. In general, our results demonstrate that intragenomic recombination has played a significant role in the evolution of the studied tpr genes and emphasize that efforts to infer evolutionary history of the treponemes can be complicated if past recombination events are not recognized.
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.
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