Sequence variability of BamA and FadL candidate vaccinogens suggests divergent evolutionary paths of <i>Treponema pallidum</i> outer membrane proteins

Knowledge of <i>Treponema pallidum</i> subspecies <i>pallidum</i> (<i>TPA</i>) outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as the design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of <i>TPA</i> BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore <i>TPA</i> strain diversity, we mapped the variability of BamA and FadL orthologs in 186 <i>TPA</i> strains from Malawi, China, and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols- and three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the <i>TPA</i> strains, effectively segregating within clades and largely clustering by subclades. Finally, we noted that BamA and FadL candidate ECL vaccinogens, previously shown to elicit antibodies that kill treponemes during <i>in vitro</i> cultivation, are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing the selection of vaccine targets.IMPORTANCESyphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of <i>TPA</i> OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six <i>TPA</i> OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of <i>TPA</i>. Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.