In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.

Abstract. Treponema pallidum ssp. pallidum (TPA) causes over 10 million new cases of syphilis worldwide whereas T. pallidum ssp. pertenue (TPE), the causative agent of yaws, affects about 2.5 million people. Although penicillin remains the drug of choice in the treatment of syphilis, in penicillin-allergic patients, macrolides have been used in this indication since the 1950s. Failures of macrolides in syphilis treatment have been well documented in the literature and since 2000, there has been a dramatic increase in a number of clinical samples with macrolide-resistant TPA. Scarce data regarding the genetics of macrolide-resistant mutations in TPA suggest that although macrolide-resistance mutations have emerged independently several times, the increase in the proportion of TPA strains resistant to macrolides is mainly due to the spread of resistant strains, especially in developed countries. The emergence of macrolide resistance in TPA appears to require a two-step process including either A2058G or A2059G mutation in one copy of the 23S rRNA gene and a subsequent gene conversion unification of both rRNA genes. Given the enormous genetic similarity that was recently revealed between TPA and TPE strains, there is a low but reasonable risk of emergence and spread of macrolide-resistant yaws strains following azithromycin treatment.

1 strains provides insights into the evolution of Treponema pallidum subsp. 2 pallidum, the syphilis spirochete 3 4 ABSTRACT 28In recent years, considerable progress has been made in topologically and 29 functionally characterizing integral outer membrane proteins (OMPs) of Treponema 30 pallidum subspecies pallidum (TPA), the syphilis spirochete, and identifying its surface-31 exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are 32 known to be highly variable. We examined the sequence diversity of β-barrel-encoding 33 regions of tprC, tprD, and bamA, in 31 specimens from Cali, Colombia; San Francisco, 34 California; and the Czech Republic and compared them to allelic variants in the 41 35 reference genomes in the NCBI database. To establish a phylogenetic framework, we 36 used tp0548 genotyping and tp0558 sequences to assign strains to the Nichols or SS14 37 clades. We found that (i) β-barrels in clinical strains could be grouped according to 38 allelic variants in TPA reference genomes; (ii) for all three OMP loci, clinical strains 39 within the Nichols or SS14 clades often harbored β-barrel variants that differed from the 40 Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in 41 predicted extracellular loops containing B-cell epitopes. Based upon structural models, 42 non-conservative amino acid substitutions in predicted transmembrane -strands of 43 TprC and TprD2 could give rise to functional differences in their porin channels. OMP 44 profiles of some clinical strains were mosaics of different reference strains and did not 45 correlate with results from enhanced molecular typing. Our observations suggest that 46 human host selection pressures drive TPA OMP diversity and that genetic exchange 47 IMPORTANCE 51Despite recent progress characterizing outer membrane proteins (OMPs) of 52 Treponema pallidum (TPA), little is known about how their surface-exposed, β-barrel-53 forming domains vary among strains circulating within high-risk populations. In this 54 study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and 55 bamA, in TPA from a large number of patient specimens from geographically disparate 56 sites were examined. Structural models predict that sequence variation within β-barrel 57 domains occurred predominantly within predicted extracellular loops. Amino acid 58 substitutions in predicted transmembrane strands that could potentially affect porin 59 channel function also were noted. Our findings suggest that selection pressures exerted 60 by human populations drive TPA OMP diversity and that recombination at OMP loci 61 contributes to the evolutionary biology of syphilis spirochetes. These results also set the 62 stage for topology-based analysis of antibody responses that promote clearance of TPA 63 and frame strategies for vaccine development based upon conserved OMP extracellular 64 loops. 65 66 67 After years of steady decline during the 1990s, syphilis, a sexually transmitted 68 infection caused by...