Genetic Profiling of the Full-Length Gene in Patients with Primary and Secondary Syphilis.

TprK antigenic variation is acknowledged as an important strategy developed by Treponema pallidum to achieve immune evasion. Previous studies applied short-read sequencing to explore gene sequence diversity in clinical samples; however, due to the limitations of short-read sequencing, it was difficult to determine the linkage between the seven V regions, and crucial information about full-length variants was lost. Although two recent studies explored complete gene profiles in natural human syphilis infection, there are still too few profiled full-length variants among clinical T. pallidum isolates to fully understand the characteristics of TprK coding diversity. Here, Pacific Biosciences (PacBio) long-read sequencing was applied to examine the diversity of full-length variants in 21 clinical T. pallidum isolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. A total of 398 high-confidence full-length sequences, which presented remarkable sequence heterogeneity, were found. However, these full-length variants exhibited limited variation in length and GC content, showing 24 length types and average GC content of 51.5 ± 0.42% and 51.6 ± 0.26% for primary and secondary syphilis samples, respectively. Additionally, the combined patterns of mutated V regions generating new variants were obviously different in primary and secondary syphilis samples. The diversity of gene sequences in primary syphilis samples may represent the underlying variability of the bacterium; conversely, the variability of the gene in secondary syphilis samples may more accurately reflect how T. pallidum escapes host immune clearance. These data highlight the gene as an important coding gene that shows conflicting genetic characteristics but underlies the persistence of spirochete infection. The resurgence of syphilis in both low- and high-income countries has attracted attention, and persistent infection by the pathogen has long been a research focus. The gene, encoding the hypervariable outer membrane protein, is thought to be responsible for pathogen immune evasion and persistent infection. Here, PacBio long-read sequencing was applied to examine the diversity of full-length variants in 21 clinical T. pallidum isolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. The results showed that the sequences of the gene were remarkably heterogeneous; however, the sequences presented limited variation in length and GC content. The investigation of the combined patterns of the V regions allowed us to gain insight into the features of the gene generating new variants at different clinical stages. The findings of this study will be helpful for further exploration of the pathogenesis of syphilis.