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Syphilis, caused by Treponema pallidum subsp. pallidum, is a chronic disease characterized by periods of activity and latency, with clearance of early lesions but persistence of infection. The mechanisms T. pallidum uses to persist in humans are still not known, but it is possible that alterations in surface proteins will be involved because of their interaction with the host and their visibility to the immune response.

A family of 12 tpr genes is contained in the T. pallidum subsp. pallidum Nichols strain genome (7), some of which code for candidate surface-exposed proteins (3, 7, 12, 13). The tpr genes of T. pallidum are of interest for a variety of reasons. Their gene products are homologous to the Msp proteins of Treponema denticola, which have been implicated in cell attachment and porin function (6, 11). Anti-TprK antibody has been shown to opsonize T. pallidum Nichols strain for phagocytosis (3); thus, TprK may be exposed at the cell surface. Immunization with TprK is partially protective against challenge with T. pallidum Nichols strain (3), suggesting some tpr gene products are a focus of the protective immune response. Finally, the variable nature of the tpr genes suggests a role in immune evasion and persistence if different tpr genes are sequentially expressed. The 12 tpr gene products can be categorized into three subfamilies (3). Subfamilies I (TprCDFI) and II (TprEGJ) have conserved amino- and carboxyl-terminal sequences, but variable central amino acid sequences (3). Subfamily III Tpr proteins (TprABHKL) have scattered variable and conserved sequences throughout their length (3).

A novel tpr gene was discovered. Multiple tprK alleles have been found in recent isolates of T. pallidum (5), in contrast to the single tprK allele identified in the laboratory-adapted Nichols strain (7). Because of our interest in tpr heterogeneity, we compared tpr gene sequences from other T. pallidum isolates to those from the Nichols strain. For example, genomic DNA from the T. pallidum subsp. pallidum Mexico A isolate was used as a template for PCR with primers A and B (Table 1), which are complementary to conserved regions flanking the central variable domains of tpr subfamilies I and II. The rabbit propagation, sources of the treponeme isolates, extraction of genomic DNA, and PCR conditions have been described previously (2-5). One of the amplicons was homologous to subfamily I (tprCDFI) at the conserved 5' and 3' ends, yet distinct from all of the subfamily I genes in much of the variable region (GenBank accession no. AF187953). This novel tpr was also found in the T. pallidum subsp. pallidum Bal-3 isolate (GenBank accession no. AF187952).

The novel tpr gene occupies the tprD locus, and thus was termed tprD2. In order to localize the novel tpr gene in the genome, inverse PCR was used to amplify a fragment of genomic DNA containing the 5' portion of the novel tpr gene and the 5' flanking DNA. Genomic Bal-3 DNA (100 ng) was digested with Sau3A1 and ligated with T4 DNA ligase (New England Biolabs, Beverly, Mass.) in a 120-µl volume, such that circles were likely to be formed. This was used as a template in a PCR with primers C and G (Table 1 and Fig. 1) and yielded a 1.7-kb amplicon. Sequencing of this amplicon demonstrated that the novel tpr gene is flanked at the 5' end by DNA with almost complete identity to the TP0132 and TP0133 genes, which are at the 5' flanking end of tprD in the T. pallidum Nichols genome (Fig. 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Diagram of the tprD locus. Three different alleles of tprD (tprD, tprD2, and tprD3) are present in the tprD locus in the strains examined. The regions denoted as arrows and with TP or tpr are predicted coding regions with putative start codons at the beginning of the arrow and putative stop codons at the point of the arrow. IGR, intergenic region. PCR products are shown as thin lines, and the primers used to produce the products are shown as arrowheads. The different shading patterns and lines within the tprD, tprD2, and tprD3 alleles demonstrate the differences between the alleles.

View larger version (81K): [in this window] [in a new window]   FIG. 2.   Amino acid sequence alignments of tprD, tprD2, and tprD3. The alignments of the predicted amino acid sequences of the tprD, tprD2, and tprD3 alleles are shown.

tprD2 was identified in about half of the Treponema isolates. Primers C and D (Table 1) were designed to amplify a tprD2-specific 273-bp amplicon (Fig. 1) to test for the presence of tprD2 in a variety of Treponema isolates. We examined 12 isolates of T. pallidum subsp. pallidum, 4 isolates of T. pallidum subsp. pertenue (yaws spirochetes), 1 isolate of T. pallidum subsp. endemicum (endemic syphilis spirochete), the Simian isolate (primate spirochete [8]), and 2 isolates of Treponema paraluiscuniculi (rabbit venereal spirochetes) (Table 2). tprD2 was detected in 7 of 12 T. pallidum subsp. pallidum genomes and in 7 of 8 of the non-syphilis treponeme genomes (Table 2). Primers specific for tprA (primers E and F, Table 1) gave the predicted 315-bp amplicon with each of these isolates, demonstrating that the DNA from each treponeme isolate was intact and amplifiable (not shown). Rabbit DNA, a likely contaminant, did not give an amplicon with these sets of primers. Thus, it appears that the tprD2 allele is present in about half of the T. pallidum subsp. pallidum isolates tested, but not the Nichols strain that was used for the genome sequencing project (7).

Humans with syphilis make antibodies to TprD2-specific peptide. An amplicon encoding a 90-amino-acid peptide (amino acids 301 to 391 in Fig. 2) unique to the predicted TprD2 protein was amplified (primers C and D, Table 1 and Fig. 1), expressed as a six-histidine fusion protein, and purified (TprD2-specific peptide) (3). This TprD2-specific peptide had no homology to any of the other predicted tpr gene products or homology to any of the predicted open reading frames of the T. pallidum Nichols strain genome.

View larger version (62K): [in this window] [in a new window]   FIG. 3.   Western blots demonstrating antibodies in sera from persons with secondary syphilis react with the TprD2-specific peptide. Shown are immunoblots with the 14-kDa TprD2-specific recombinant peptide from a representative experiment. The immunoblots were reacted with sera from two uninfected controls (U), five persons with secondary syphilis (noted by no. 2), and two persons with late latent syphilis (LL). Antibody reactivity to the tprD2-specific peptide is seen with the sera from three of five persons with secondary syphilis. Shown to the right are the positions of the molecular mass markers.