Comparative Genomics and Full-Length TprK Profiling ofTreponema pallidumsubsp.pallidumReinfection

Abstract

<jats:title>Abstract</jats:title><jats:p>Developing a vaccine against <jats:italic>Treponema pallidum</jats:italic> subspecies <jats:italic>pallidum</jats:italic>, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro <jats:italic>T. pallidum</jats:italic> culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of <jats:italic>T. pallidum</jats:italic> reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length <jats:italic>tprK</jats:italic> profiling of two <jats:italic>T. pallidum</jats:italic> isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable <jats:italic>tprK</jats:italic> gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length <jats:italic>tprK</jats:italic> deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each <jats:italic>tprK</jats:italic> variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of <jats:italic>T. pallidum</jats:italic> to successfully reinfect individuals, even when they have been infected with the organism multiple times.</jats:p><jats:sec><jats:title>Author Summary</jats:title><jats:p>The causative agent of syphilis, <jats:italic>Treponema pallidum</jats:italic> subspecies <jats:italic>pallidum</jats:italic>, is capable of repeat infections in people, suggesting that the human immune response does not develop sufficiently broad or long-lasting immunity to cover treponemal diversity. Here, we examined the genomes from two blood-derived isolates of <jats:italic>T. pallidum</jats:italic> derived 6 years apart from a patient who had syphilis four times during the same period to understand the genetic basis of reinfection. We found a paucity of coding changes across the genome outside of the highly variable <jats:italic>tprK</jats:italic> gene. Using deep profiling of the full-length <jats:italic>tprK</jats:italic> gene, we found surprisingly that the two isolates did not share any full-length TprK sequences.</jats:p></jats:sec>

Estimation of Full-Length TprK Diversity inTreponema pallidumsubspeciespallidum

“…In a previous investigation, we profiled TprK in two T. pallidum strains collected from a single patient and amplified by two passages of strains in New Zealand white rabbits [24]. To assess the impact of the additional passage through rabbits on TprK, we compared the number of unique variants and diversity across the seven V regions identified from the 13 Italian T. pallidum strains and our two previously profiled strains.…”

“…The treponemal load of each sample was measured by qPCR as previously described [24]. Briefly, a portion of tp47 was amplified using 14.33 µL of 2x QuantiTect multiplex PCR mix, 0.65 µL of 2x QuantiTect multiplex PCR mix with ROX, 0.03 unit of UNG and the following primers 5′-CAA GTA CGA GGG GAA CAT CGA T-3′ and 5′-TGA TCG CTG ACA AGC TTA GG-3′.…”

“…PCR amplification of tprK was conducted using previously described conditions [24] and tprK -specific primers appended to 16 bp Pacbio barcodes (Table S1). The resulting 1.7kb product was cleaned using 0.6x volumes of AMPure XP beads (Beckman-Coulter).…”

“…Because of the slight differences in coverage, we required a minimum of 5 reads of support for a given variable region amino acid sequence from the Xiamen samples, while for the Italian samples we used a minimum of 10 reads. We additionally included short-read sequencing data from 2 T. pallidum strains passaged in rabbits, which we profiled in a previous investigation [24], in our analysis. Similar to the Italian strains, we required each unique identified variable region sequence in these strains to be supported by a minimum of 10 sequencing reads and present at or above a frequency of 0.2%.…”

“…Previous studies to evaluate TprK variability within T. pallidum strains have sequenced of a limited number of TprK clones, failed to resolve linkage between variable regions, or been conducted on strains passed through rabbits [16,18–20] [24]. As a result, no studies to date have adequately profiled TprK within T. pallidum during natural infection in the human host.…”