Antigenic variation of TprK facilitates development of secondary syphilis

Abstract

Although primary syphilis lesions heal spontaneously, the infection is chronic, with subsequent clinical stages. Healing of the primary chancre occurs as antibodies against outer membrane antigens facilitate opsonophagocytosis of the bacteria by activated macrophages. TprK is an outer membrane protein that undergoes antigenic variation at 7 variable regions, and variants are selected by immune pressure. We hypothesized that individual TprK variants escape immune clearance and seed new disseminated lesions to cause secondary syphilis. As in human syphilis, infected rabbits may develop disseminated secondary skin lesions. This study explores the nature of secondary syphilis, specifically, the contribution of antigenic variation to the development of secondary lesions. Our data from the rabbit model show that the odds of secondary lesions containing predominately TprK variant treponemes is 3.3 times higher than the odds of finding TprK variants in disseminated primary lesions (odds ratio [OR] = 3.3 [95% confidence interval {CI}, 0.98 to 11.0]; P = 0.055) and that 96% of TprK variant secondary lesions are likely seeded by single treponemes. Analysis of antibody responses demonstrates significantly higher antibody titers to tprK variable region sequences found in the inoculum compared to reactivity to tprK variant sequences found in newly arising secondary lesions. This suggests that tprK variants escape the initial immune response raised against the V regions expressed in the inoculum. These data further support a role for TprK in immune evasion and suggest that the ability of TprK variants to persist despite a robust immune response is instrumental in the development of later stages of syphilis.

FIG 1

Examples of TprK V7 sequences among secondary lesions compared to the inoculum. For each lesion (identified by number on the left), DNA sequences were obtained from 10 Escherichia coli clones containing the tprK ORF. Note that the sequence variation in V7 is seen as insertions/deletions and base changes (numbers at the top indicate base pair positions). The identity, or near identity, of the 10 sequences within each lesion supports the single-cell origin of the lesion.

FIG 2

Phylogenetic analysis of full-ORF tprK DNA sequences from secondary lesions. A representative example of phylogenetic relationships (a maximum-parsimony cladogram) among the inoculum and the individual secondary lesions (identified by rabbit number and lesion letter) that developed in rabbit 7901 is shown. Lesion 7901B did not contain amplifiable treponemes. The clusters enclosed by shaded ovals correspond to the sequences from 10 plasmid clones per lesion. Bootstrap values of the major branches are shown, demonstrating separation of the lesion sequences from the inoculum sequence. Note that the sequences for each lesion are in tight clusters, consistent with a single-cell origin for each lesion.

FIG 3

TprK variants found in secondary lesions escape antibody binding. (A) Antibody binding (ELISA) of antiserum (collected at the time of development of secondary lesions [60 days postinfection]) from rabbit 7912 against synthetic peptides representing V region sequences identified in the inoculum (blue bars) and in two secondary lesions (red and yellow bars) that arose in the rabbit. Antibody reactivity is seen to the predominant sequences of the inoculum V2, V4, V6, and V7 regions, but significantly lower reactivity was detected to the V region sequences found in the secondary lesions. Note also that there is significantly lower antibody reactivity to the two minority V6 sequences that were present in the inoculum (also in blue). Sera from all rabbits with secondary syphilis were similarly tested and showed the same pattern of reactivity to the majority inoculum sequences, but not to the minor inoculum sequences or the variant secondary-lesion sequences. (B) Mean (±standard error [SE]) ELISA results for antisera (collected at the time of lesion appearance [mean, 22 days postinfection]) from rabbits with disseminated primary syphilis (n = 5 rabbits) showing lack of antibody reactivity directed to the V region sequences from the inoculum. *, two-tailed, paired Student t test; P < 0.001 compared to antibody binding to the peptide representing the majority sequences identified in the inoculum. Abs 405 nm, absorbance at 405 nm.