Analysis of <i>Treponema pallidum</i> subsp. <i>pallidum</i> predicted outer membrane proteins (OMPeomes) in 21 clinical samples: variant sequences are predominantly surface-exposed

The incidence of syphilis, a sexually transmitted disease caused by the Treponema pallidum subsp. pallidum (TPA), has been surging globally despite effective antibiotic therapy. A new strategy for syphilis control is the development of a multi-component syphilis vaccine with global efficacy, which requires the identification of surface-exposed candidate vaccinogens and the determination of their antigenic diversity within circulating TPA strains. To improve the quality of sequences from repetitive and paralogous regions of the TPA genome, we have developed a sequencing scheme that allows amplification and long-read sequencing of 25 targets encoding TPA proteins including 15 outer membrane proteins. We tested this approach on a set of 21 clinical TPA strains, mostly of European origin preselected by MLST typing. A total of 462 (88%) of 525 amplicons were sequenced. Of 58 new alleles identified in comparison to the SS14 and Nichols TPA reference strains, the majority encoded new protein sequences ( n = 55; 94.8%). The 55 variant protein sequences were encoded by 99 individual TPA loci, where single amino acid replacements occurred most frequently ( n = 50), followed by replacements of two to three amino acids ( n = 35) and differences comprising four or more residues ( n = 14); the latter included six intra-strain recombination events. Most differences were localized to predicted surface-exposed regions, consistent with adaptive evolution of bacterial determinants that function at the host-pathogen interface. Clinical strains having the same allelic profiles from different localities differed in several loci, suggesting that geographical origin significantly contributes to genetic diversity of circulating strains. IMPORTANCE Our findings underscore the importance of analyzing TPA clinical samples isolated from diverse geographical regions in order to understand TPA OMP variability.