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.

Treponema pallidum subsp. pallidum with an Artificially impaired TprK antigenic variation system is attenuated in the Rabbit model of syphilis

“…Raw reads were adapter-trimmed and filtered for quality scores above Q20 using Trimmomatic v0.39 [ 54 ], followed by merging the paired end reads using BBmerge ( https://www.osti.gov/biblio/1241166 ) in default parameters. Variable site sequences were identified using custom python and R scripts [ 22 ], and those containing frameshifts or nonsense mutations in the translated sequences removed. Replicate samples yielding a minimum of 2,000 reads in all V regions in both replicates were kept for downstream analysis, corresponding to input merged reads ranging between 89182–5746195.…”

“…Aside from sequence variability and potential for generation of quintillions of unique sequences, the role of TprK in immune evasion and pathogen persistence is supported by the evidence that a) sequence diversity accumulates over time during experimental infection [ 18 ], b) infection-induced antibodies preferentially target the TprK V regions rather than the predicted β-barrel scaffolding of the protein [ 19 ], c) sequence variation abrogates binding of antibodies raised to antigenically different V regions [ 20 ], d) TprK variants accumulate more rapidly in immunocompetent hosts compared to immunosuppressed rabbits as the result of immune selection [ 21 , 22 ] and, lastly, e) TprK sequences found in disseminated secondary lesions in the rabbit model differ from those in the original inoculum, supporting that secondary lesions contain treponemes that avoided the initial round of immune clearance associated with the resolution of the primary lesion(s) [ 23 ].…”

Strategies for syphilis vaccine development

“…Our many attempts to ablate the OMPencoding tprK open reading frame, however, were constantly unsuccessful, which led to the hypothesis that tprK is an essential gene of the syphilis spirochete. This hypothesis is also indirectly supported by the evidence that high-throughput tprK sequencing never yielded a variant carrying an early termination due to the introduction of a stop codon or a frameshift mutation, despite the continuous recombination events that occur to create variability in this gene (45)(46)(47) . A vaccine design based on a surface-exposed antigen that mediates crucial functions in T. pallidum biology could be more valuable than a design based on a nonessential gene, whose function could be redundant.…”

Clonal isolates of Treponema pallidum subsp. pallidum Nichols provide evidence for the occurrence of microevolution during experimental rabbit infection and in vitro culture

“…The spirochetes are transmitted by contact of lesion exudates with epidermal or mucosal surfaces (and by transplacental passage in congenital syphilis); they give rise to local lesions and often disseminate to distant tissues. These infections may last and cause manifestations for years to decades, reflecting the expression of immune evasion mechanisms, including the paucity of surface proteins and antigenic variation of the protein TprK through a gene conversion mechanism [9][10][11][12][13][14].…”