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

Abstract

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.

FIG. 1

Orientation and nucleotide position of the different primers used for PCR and sequencing of the central regions of the tprK ORF as well as the complete ORF and its flanking regions. Solid lines represent the ORF of the tprK gene. Primer positions are as follows: F5-S, base positions 975967 through 975987 upstream of the tprK start codon; FW-S, base positions 975809 to 975833; 9V-S, base positions 975629 to 975652 in the tprK ORF; tprK-S, base positions 975162 to 975187 in the tprK ORF; tprK-As, base positions 974778 to 974800 in the tprK ORF; 9V-As, base positions 974708 to 974730 in the tprK ORF; FW-As, base positions 974319 to 974341 in the tprK ORF; and F3-As, base positions 973922 to 973943 downstream of the tprK stop codon.

FIG. 2

High-resolution ethidium bromide agarose gel showing amplicons of T. pallidum isolates obtained with the short-range primers (tprK-S and tprK-As) which amplify a central region in a large hydrophilic domain of the TprK antigen. Amplicons vary in the number of bands and sizes.

FIG. 3

Alignment of the TprK internal region predicted amino acid sequences of T. pallidum isolates obtained with the tprK-S and tprK-As primers showing highly conserved regions and three variable domains. Shaded areas indicate sequence identity, and broken lines indicate gaps in the alignment. NicholsGen, Nichols strain sequence as reported in the T. pallidum genome sequence; NicholsSea, sequence of the Nichols strain maintained in our laboratory. The street isolates shown in this alignment are Bal 7, Bal 73-1, and Sea 81-4 listed in Table 1. The sequences in Fig. 3 to 5 are indicated by isolate name, followed by the clone designation. For example, Bal 73-1.306 is the sequence from clone 306 of the Bal 73-1 isolate.

FIG. 4

Alignment of the predicted amino acid sequences of complete ORFs identified in the Nichols tprK locus in five T. pallidum isolates, showing seven regions of heterogeneity (V₁ to V₇). Shaded areas indicate sequence identity, and broken line show the gaps in the alignment. NicholsGen, Nichols strain sequence as reported in the T. pallidum genome sequence; NicholsSea, sequence of the Nichols strain maintained in our laboratory. Bal 7, Bal 73-1, and Sea 81-4 are street isolates (Table 1). Additional numbers indicate the clone designations from each isolate. All sequences are from amplicons obtained with the F5-S and F3-As primers which bind the tprK flanking regions; therefore, no primer binding sites are included in this alignment.

FIG. 5

DNA alignment of the last 16 nucleotides of the tprK ORF and its 3′-flanking region from the Bal 7, Bal 73-1, and Sea 81-4 street isolates and from the Nichols strain (NicholsSea, strain maintained in our laboratory; NicholsGen, genome Nichols strain). Single underline indicates the tprK stop codon. Open and hatched boxes indicate the two putative transcription termination hairpin structures. Broken lines indicate a sequence deletion of 67 nucleotides in the NicholsSea isolate and the Bal 7 isolate. Sequences shown are the complement of the corresponding regions in the T. pallidum genome.