Molecular evolution of the tprC, D, I, K, G, and J genes in the pathogenic genus Treponema.

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.

Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92.

By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%-100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.

The tprK gene is heterogeneous among Treponema pallidum strains and has multiple alleles.

We have previously shown that the TprK antigen of T. pallidum, Nichols strain, is predominantly expressed in treponemes obtained 10 days after infection and that the hydrophilic domain of TprK is a target of opsonic antibodies and confers significant protection against homologous challenge. The T. pallidum genome sequence reported the presence of a single copy of the tprK gene in the Nichols strain. In the present study we demonstrate size heterogeneity in the central portions of the TprK hydrophilic domains of 14 treponemal isolates. Sequence analysis of the central domains and the complete open reading frames (ORFs) of the tprK genes confirms this heterogeneity. Further, multiple tprK sequences were found in the Nichols-defined tprK locus in three isolates (Sea 81-4, Bal 7, and Bal 73-1). In contrast, only a single tprK sequence could be identified in this locus in the Nichols strain. Alignment of the DNA and deduced amino acid sequences of the whole tprK ORFs shows the presence of seven discrete variable domains flanked by highly conserved regions. We hypothesize that these heterogeneous regions may be involved in antigenic heterogeneity and, in particular, evasion of the immune response. The presence of different tprK alleles in the tprK locus strongly suggests the existence of genetically different subpopulations within treponemal isolates.