Profile of the tprK gene in primary syphilis patients based on next-generation sequencing

Abstract

BackgroundThe highly variable tprK gene of Treponema pallidum has been acknowledged to be the cause of persistent infection. Previous studies mainly focused on the heterogeneity in tprK in propagated strains using a clone-based Sanger approach. Few studies have investigated tprK directly from clinical samples using deep sequencing.\n\nMethods/Principal findingsWe conducted a comprehensive analysis of 14 primary syphilis clinical isolates of T. pallidum via next-generation sequencing to gain better insight into the profile of tprK in primary syphilis patients. Our results based on primary syphilis clinical samples showed that there was a mixture of distinct sequences within each V region of tprK. Except for the predominant sequence for each region as previously reported using the clone-based Sanger approach, there were many minor variants of all strains that were mainly observed at a frequency of 1-5%. Interestingly, the identified distinct sequences within the regions were variable in length and differed only by 3 bp or multiples of 3 bp. In addition, amino acid sequence consistency within each region was found between the 14 strains. Among the regions, the sequence IASDGGAIKH in V1 and the sequence DVGHKKENAANVNGTVGA in V4 showed a high stability of inter-strain redundancy.\n\nConclusionsThe seven V regions of the tprK gene in primary syphilis infection demonstrated high diversity; they generally contained a high proportion sequence and numerous low-frequency minor variants, most of which are far below the detection limit of Sanger sequencing. The rampant variation in each region was regulated by a strict gene conversion mechanism that maintained the length difference to 3 bp or multiples of 3 bp. The highly stable sequence of inter-strain redundancy may indicate that the sequences play a critical role in T. pallidum virulence. These highly stable peptides are also likely to be potential targets for vaccine development.\n\nAuthor summaryVariations in tprK have been acknowledged to be the major contributors to persistent Treponema pallidum infections. Previous studies were based on the clone-based Sanger approach, and most of them were performed in propagated strains using rabbits, which could not reflect the actual heterogeneous characteristics of tprK in vivo. In the present study, we employed next-generation sequencing (NGS) to explore the profile of tprK directly from 14 patients with primary syphilis. Our results showed a mixture of distinct sequences within each V region of tprK in these clinical samples. First, the length of identified distinct sequences within the region was variable, which differed by only 3 bp or multiples of 3 bp. Then, among the mixtures, a predominant sequence was usually observed for each region, and the remaining minor variants were mainly observed at a frequency of 1-5%. In addition, there was a scenario of amino acid sequence consistency within the regions between the 14 primary syphilis strains. The identification of the profile of tprK in the context of human primary syphilis infection contributes to further exploration of the pathogenesis of syphilis.