Treponema pallidum subsp. pallidum genetic population structure and relevance to syphilis prevention and treatment.

Syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (TPA), is resurging globally, particularly in low- and middle-income countries. 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 β-barrel scaffolds. Macrolide resistance and reduced beta-lactam susceptibility marker prevalence varied by lineage and geographical region. These findings refine our understanding of TPA's 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 for antimicrobial resistance monitoring. Despite increasing syphilis rates worldwide, genomic data from its causative agent Treponema pallidum subsp. pallidum have largely originated from a small number of high-income countries. This study significantly expands our understanding of TPA genomic diversity by sampling across underrepresented low- and middle-income countries (LMICs) in Africa, Asia and South America. We analyze 1,707 genomes, including 298 newly sequenced from 11 LIMCs, and identify novel subpopulations, lineage-specific variation in outer membrane proteins, and geographic differences in antimicrobial resistance markers. These results illustrate how sampling from understudied regions reveals previously unappreciated, geographically structured diversity, improving our understanding of TPA population structure and informing the development of globally relevant vaccines, treatment strategies, and diagnostic tools.