Genomic analysis of 1,707 syphilis genomes reveals lineage-specific diversity and resistance markers across 11 countries

Syphilis, caused by the spirochete Treponema pallidum subsp. Pallidum (TPA), is rapidly resurging globally, particularly in low- and middle-income countries where the burden is increasingly concentrated. However, TPA genomic diversity and population structure in these settings remain poorly characterized. We investigated the global genetic diversity of syphilis spirochetes, sequencing 298 new TPA genomes from 11 countries across five continents, including underrepresented areas such as Argentina, Colombia, Malawi, Sri Lanka, and Vietnam. Combined with 1,409 public genomes, our dataset comprised 1,707 genomes. Hierarchical clustering identified six Nichols and five SS14-lineage subpopulations, with distinct subpopulations concentrated in Africa, East Asia, and the Americas, as well as previously unrecognized diversification within the globally dominant SS14 lineage. Concordance analysis showed that widely used multilocus sequencing typing methods recapitulate major Nichols-lineage subpopulations but have reduced discriminatory power for the SS14 lineage. Genome-wide Fixation index scans and targeted analyses of genes encoding outer membrane proteins prioritized for vaccine development demonstrated lineage- and subpopulation-specific patterns of genetic structure and selection. We observed strong diversifying selection acting on cell envelope assembly factors (BamA, LptD), selected FadL-like transporters, members of the T. pallidum repeat (Tpr) family, and efflux-associated outer membrane factors, alongside strictly conserved {beta}-barrel scaffolds. Macrolide resistance and reduced beta-lactam susceptibility marker prevalence varied by lineage and geographical region. These findings refine our understanding of TPA genetic diversity, delineate heterogeneous evolutionary trajectories across key vaccine-relevant loci, and underscore the importance of geographically representative genomic analyses to inform syphilis vaccine design and antimicrobial resistance monitoring.