Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure

Abstract

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.

Fig 1. Anatomy of the tprK gene and mechanism of antigenic variation.

(A) tprK is composed of seven discrete variable regions (V1 –V7), color-coded here. (B) Flanking the tprD ORF (not to scale) are donor site segments used to generate unique V region sequences, again color-coded by their use in their respective variable regions. 51 of the known 53 donor sites are present in the 3’ flanking region. The segments used can be complete or partial, and vary in number for each V region sequence. They are combined through non-reciprocal segmented gene conversion to create antigenic variation.

Fig 2. Study design for sample acquisition from (A) immunosuppressed vs. immunocompetent rabbits and (B) in vitro propagated treponemes vs. control.

(A) Five immunocompetent rabbits and five immunosuppressed rabbits were infected with inoculum at Week 0. Harvesting of punch biopsies from each rabbit was done weekly for 5 weeks. Samples were analyzed for treponemal load by qPCR and tprK sequence by next-generation sequencing. (B) Glycerol stock of T. pallidum Chicago C was thawed at Week -2, passaged in vitro at Week -1 and again at Week 0, at which point treponemes were 1) inoculated intradermally into a control rabbit, and 2) seeded into two sets of weekly-passaged cultured samples for 7 weeks. Samples were collected weekly from punch biopsies of the rabbit and from cell culture, then analyzed for treponemal load and tprK sequence.

Fig 3. Log copy numbers for samples passaged in (A) immunosuppressed vs. immunocompetent rabbits and (B) immunocompetent rabbit vs. in vitro culture system.

Each unique sample is represented by either a dot or a cross, where a cross indicates that we were unable to successfully recover sequence from the sample. Lines show the mean normalized treponemal load, as quantified by the log ratio of tp0574 copies to CFTR copies and are grouped and color-coded by passage type.

Fig 4. Longitudinal allelic diversity of samples passaged in (A) immunosuppressed vs. immunocompetent rabbits and (B) immunocompetent rabbit vs. in vitro culture system.

Data are separated by variable region. Dots represent the mean Pielou’s evenness score across different color-coded groups, with lines marking the standard deviation.

Fig 5. Comparison of number of unique V region alleles detected in samples passaged in immunosuppressed rabbits and in culture.

X-axis depicts weeks post-infection. Dots represent the mean number of unique variants at that time point, with lines demonstrating standard deviation.

Fig 6. Similar relative frequencies (RFs) of tprK variable region alleles are observed across culture replicates.

(A) Comparison of relative frequencies of alleles between two sets of culture passages. Dots represent unique variable region sequences of varying timepoints, color-coded by region. Dotted gray line plots Y = X. (B) Comparison of relative frequencies of all novel alleles not in inoculum between two culture replicates, color-coded by variable region. (C) Relative frequencies of alleles between both culture replicates, at over 70%. Data are grouped by variable region and color coded by week. (D) Correlation between relative frequencies of alleles in culture replicates at low frequencies over time. Dots represent unique variable region sequences between timepoints, with corresponding regression lines plotted. Both are color-coded by week.

Fig 7. Comparison between culture replicates of individual novel V region alleles absent in inoculum that reached ≥0.4% relative frequency at any timepoint.

Unique alleles are labeled above each plot with their variable region and amino acid sequence. Lines sequentially connect each timepoint and are color-coded by week.

Fig 8. Donor site contributions for novel tprK V region alleles present in any timepoint above 0.4% relative frequency.

Selected variable regions with newly arising tprK alleles are depicted, with the clonal sequence at the top and highlighted in salmon, followed by the next highest sequence in relative frequency, and then novel alleles absent from inoculum, demarcated by red stars. Donor site segments are illustrated with color-coded arrows above their respective tprK sequences for each variable region. White donor site segments in V1 are segments that are present in all V1 donor sites. The translated amino acid sequences are at the top left of each allele. Range of relative frequencies of each allele are reported, with week numbers and respective relative frequencies.