Comparative genomics and full-length Tprk profiling of Treponema pallidum subsp. pallidum reinfection

Abstract

Developing a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum 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 T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, fulllength tprK profiling of two T. pallidum 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 tprK 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 tprK 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 tprK 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 T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times.

Estimation of Full-Length TprK Diversity in Treponema pallidum subsp.pallidum

“…In a previous investigation, we profiled TprK in two T. pallidum subsp. pallidum isolates (UW-148B and UW-148B2) collected from a single patient and amplified by two passages of strains in New Zealand White rabbits ( 23 ). To assess the impact of the additional passage through rabbits on TprK, we compared the number of unique variants and diversity across the 7 V regions identified from the 13 Italian T. pallidum subsp.…”

“…The treponemal load of each sample was measured by quantitative PCR (qPCR) as previously described ( 23 ). Briefly, a portion of tp47 was amplified using 14.33 μl of 2× QuantiTect multiplex PCR mix, 0.65 μl of 2× 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 the high-fidelity CloneAmp polymerase (TaKaRa) and tprK -specific primers appended to 16-bp PacBio barcodes (see Table S4 in the supplemental material) with 1,000 copies of treponemal DNA input under previously described conditions ( 23 ). The resulting 1.7-kb product was cleaned using 0.6× volumes of AMPure XP beads (Beckman-Coulter).…”

“…We additionally included short-read sequencing data from 2 T. pallidum subsp. pallidum strains passaged in rabbits, which we profiled in a previous investigation ( 23 ), in our analysis. Similar to the Italian strains, we performed a technical replicate of tprK PCR and short-read sequencing and included only high-confidence variable region sequences that were present at a read count greater than 5 and a relative frequency greater than 0.1%.…”

“…Previous studies to evaluate TprK variability within T. pallidum subsp. pallidum strains have sequenced a limited number of TprK clones, failed to resolve linkage between variable regions, or been conducted on strains passed through rabbits ( 16 – 19 , 23 ). As a result, no studies to date have adequately profiled TprK within T. pallidum subsp.…”