Lineage-specific and geographic structuring of variants in genes encoding penicillin-binding proteins in Treponema pallidum

To characterize the global mutational landscape of genes encoding penicillin-binding proteins (PBPs) or β-lactam-interacting proteins in Treponema pallidum (T. pallidum), examine subspecies- and lineage-specific distribution patterns and geographic heterogeneity, and provide a reference for interpreting the potential molecular basis of reduced susceptibility to β-lactam agents. Whole-genome sequences from 737 T. pallidum genomes originating from 40 countries across six continents were analyzed using the Nichols strain (GenBank: CP004010.2) as the reference genome. Nonsynonymous variants were identified in four genes encoding penicillin-binding or β-lactam-interacting proteins: pbp1, pbp2, mrcA, and tp47. Site-level variant frequencies were calculated using site-specific callable denominators after excluding NA calls. Exact mutation-profile analyses were restricted to the 462 genomes callable at all 61 sites. Group differences were evaluated using conditional fixed-margin Monte Carlo tests based on Pearson's χ² statistic, with Benjamini-Hochberg correction for multiple comparisons; association strength was quantified using Cramér's V. A total of 61 nonsynonymous variant sites were identified. These variants were unevenly distributed and concentrated at a limited number of recurrent sites, with clear subspecies- and lineage-specific patterns. The SS14 lineage was marked by the frequent co-occurrence of pbp1 P564L and tp47 S394R, consistent with a conserved core variant profile. By contrast, mrcA A506T was associated with the Nichols lineage (V = 0.496), whereas tp47 V18A was detected in all callable T. pallidum subsp. pertenue (TPE) and T. pallidum subsp. endemicum (TEN) genomes, consistent with a shared TPE/TEN background-associated difference relative to Nichols (V = 1.000). Among the 462 complete-case genomes, 295 (63.9%) carried exact multiple-variant profiles. Profiles involving three genes were the most common, occurring in 241/462 genomes (52.2%) and predominantly in the SS14 lineage. Geographic analysis of variant-positive genomes showed substantial regional structuring, although these patterns should be interpreted in the context of subspecies and lineage composition. Europe and North America were enriched for a four-site core combination (pbp1 P564L + mrcA G708S + mrcA M625V + tp47 S394R), East Asia showed enrichment of A506V-containing pbp1-mrcA-tp47 three-gene profiles, and Africa and Oceania were characterized mainly by single-site tp47 V18A variants associated with TPE circulation. Genes encoding penicillin-binding or β-lactam-interacting proteins in T. pallidum showed lineage-specific and geographically structured nonsynonymous variation. We propose a core-backbone plus regional-accessory model, in which the SS14 lineage carries a widely distributed conserved core variant profile, whereas regional differences are shaped by additional accessory variants together with subspecies and lineage composition. This framework supports the interpretation of candidate β-lactam-interacting loci in their evolutionary and geographic context and highlights the value of continued global molecular surveillance.IMPORTANCESyphilis caused by Treponema pallidum remains a persistent global public health burden, and emerging reduced susceptibility to β-lactams threatens syphilis treatment and control. This study systematically characterizes global mutational profiles of penicillin-binding and β-lactam-interacting proteins in T. pallidum based on hundreds of worldwide genomes. It reveals distinct lineage-specific and geographically structured variant patterns and proposes a novel evolutionary variation model. These findings fill gaps in the global molecular epidemiology of T. pallidum β-lactam-related mutations and provide critical references for clinical resistance interpretation and targeted global surveillance of syphilis.