The incidence of syphilis, a sexually transmitted disease caused by the Treponema pallidum subsp. pallidum (TPA), has been surging globally despite effective antibiotic therapy. A new strategy for syphilis control is the development of a multi-component syphilis vaccine with global efficacy, which requires the identification of surface-exposed candidate vaccinogens and the determination of their antigenic diversity within circulating TPA strains. To improve the quality of sequences from repetitive and paralogous regions of the TPA genome, we have developed a sequencing scheme that allows amplification and long-read sequencing of 25 targets encoding TPA proteins including 15 outer membrane proteins. We tested this approach on a set of 21 clinical TPA strains, mostly of European origin preselected by MLST typing. A total of 462 (88%) of 525 amplicons were sequenced. Of 58 new alleles identified in comparison to the SS14 and Nichols TPA reference strains, the majority encoded new protein sequences (n = 55; 94.8%). The 55 variant protein sequences were encoded by 99 individual TPA loci, where single amino acid replacements occurred most frequently (n = 50), followed by replacements of two to three amino acids (n = 35) and differences comprising four or more residues (n = 14); the latter included six intra-strain recombination events. Most differences were localized to predicted surface-exposed regions, consistent with adaptive evolution of bacterial determinants that function at the host-pathogen interface. Clinical strains having the same allelic profiles from different localities differed in several loci, suggesting that geographical origin significantly contributes to genetic diversity of circulating strains.IMPORTANCEOur findings underscore the importance of analyzing TPA clinical samples isolated from diverse geographical regions in order to understand TPA OMP variability.

Connecticut Children's Hartford Connecticut USA

Department of Infectious Diseases Public Health Laboratory Public Health Service of Amsterdam Amsterdam the Netherlands

Department of Mycology Bacteriology Instituto de Medicina Tropical Pedro Kourí Havana Cuba

Duke University Durham North Carolina USA

INSERM Institut Cochin U1016 CNRS UMR8104 Équipe Biologie Cutané and GHU AP HP Centre Université de Paris Cité Hôpital Cochin Service de Dermatologie Vénéréologie CeGIDD CNR IST Bactériennes Paris France

Masaryk University Brno Czech Republic

UConn Health Farmington Connecticut USA

University of North Carolina at Chapel Hill Chapel Hill North Carolina USA

Zurich Institute of Forensic Medicine University of Zurich Zürich Switzerland

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Peeling RW, Mabey D, Chen X-S, Garcia PJ. 2023. Syphilis. Lancet 402:336–346. doi: 10.1016/S0140-6736(22)02348-0 PubMed DOI

Newman L, Rowley J, Vander Hoorn S, Wijesooriya NS, Unemo M, Low N, Stevens G, Gottlieb S, Kiarie J, Temmerman M. 2015. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One 10:e0143304. doi: 10.1371/journal.pone.0143304 PubMed DOI PMC

Cameron CE. 2018. Syphilis vaccine development: requirements, challenges, and opportunities. Sex Transm Dis 45:S17–S19. doi: 10.1097/OLQ.0000000000000831 PubMed DOI PMC

Ávila-Nieto C, Pedreño-López N, Mitjà O, Clotet B, Blanco J, Carrillo J. 2023. Syphilis vaccine: challenges, controversies and opportunities. Front Immunol 14:1126170. doi: 10.3389/fimmu.2023.1126170 PubMed DOI PMC

Liu A, Giacani L, Hawley KL, Cameron CE, Seña AC, Konda KA, Radolf JD, Klausner JD. 2024. New pathways in syphilis vaccine development. Sex Transm Dis 51:e49–e53. doi: 10.1097/OLQ.0000000000002050 PubMed DOI PMC

Arora N, Schuenemann VJ, Jäger G, Peltzer A, Seitz A, Herbig A, Strouhal M, Grillová L, Sánchez-Busó L, Kühnert D, et al. 2016. Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster. Nat Microbiol 2:16245. doi: 10.1038/nmicrobiol.2016.245 PubMed DOI

Pinto M, Borges V, Antelo M, Pinheiro M, Nunes A, Azevedo J, Borrego MJ, Mendonça J, Carpinteiro D, Vieira L, Gomes JP. 2016. Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation. Nat Microbiol 2:16190. doi: 10.1038/nmicrobiol.2016.190 PubMed DOI

Grillová L, Bawa T, Mikalová L, Gayet-Ageron A, Nieselt K, Strouhal M, Sednaoui P, Ferry T, Cavassini M, Lautenschlager S, Dutly F, Pla-Díaz M, Krützen M, González-Candelas F, Bagheri HC, Šmajs D, Arora N, Bosshard PP. 2018. Molecular characterization of Treponema pallidum subsp. pallidum in Switzerland and France with a new multilocus sequence typing scheme. PLoS One 13:e0200773. doi: 10.1371/journal.pone.0200773 PubMed DOI PMC

Grillová L, Oppelt J, Mikalová L, Nováková M, Giacani L, Niesnerová A, Noda AA, Mechaly AE, Pospíšilová P, Čejková D, Grange PA, Dupin N, Strnadel R, Chen M, Denham I, Arora N, Picardeau M, Weston C, Forsyth RA, Šmajs D. 2019. Directly sequenced genomes of contemporary strains of syphilis reveal recombination-driven diversity in genes encoding predicted surface-exposed antigens. Front Microbiol 10:1691. doi: 10.3389/fmicb.2019.01691 PubMed DOI PMC

Chen W, Šmajs D, Hu Y, Ke W, Pospíšilová P, Hawley KL, Caimano MJ, Radolf JD, Sena A, Tucker JD, Yang B, Juliano JJ, Zheng H, Parr JB. 2021. Analysis of Treponema pallidum strains from China using improved methods for whole-genome sequencing from primary syphilis chancres. J Infect Dis 223:848–853. doi: 10.1093/infdis/jiaa449 PubMed DOI PMC

Beale MA, Marks M, Cole MJ, Lee M-K, Pitt R, Ruis C, Balla E, Crucitti T, Ewens M, Fernández-Naval C, et al. 2021. Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis. Nat Microbiol 6:1549–1560. doi: 10.1038/s41564-021-01000-z PubMed DOI PMC

Lieberman NAP, Lin MJ, Xie H, Shrestha L, Nguyen T, Huang M-L, Haynes AM, Romeis E, Wang Q-Q, Zhang R-L, et al. 2021. Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of nichols clade strains in Madagascar. PLoS Negl Trop Dis:e0010063. doi: 10.1371/journal.pntd.0010063 PubMed DOI PMC

Taouk ML, Taiaroa G, Pasricha S, Herman S, Chow EPF, Azzatto F, Zhang B, Sia CM, Duchene S, Lee A, Higgins N, Prestedge J, Lee YW, Thomson NR, Graves B, Meumann E, Gunathilake M, Hocking JS, Bradshaw CS, Beale MA, Howden BP, Chen MY, Fairley CK, Ingle DJ, Williamson DA. 2022. Characterisation of Treponema pallidum lineages within the contemporary syphilis outbreak in Australia: a genomic epidemiological analysis. Lancet Microbe 3:e417–e426. doi: 10.1016/S2666-5247(22)00035-0 PubMed DOI

Seña AC, Matoga MM, Yang L, Lopez-Medina E, Aghakhanian F, Chen JS, Bettin EB, Caimano MJ, Chen W, Garcia-Luna JA, et al. 2024. Clinical and genomic diversity of Treponema pallidum subspecies pallidum to inform vaccine research: an international, molecular epidemiology study. Lancet Microbe 5:100871. doi: 10.1016/S2666-5247(24)00087-9 PubMed DOI PMC

Šmajs D, Strouhal M, Knauf S. 2018. Genetics of human and animal uncultivable treponemal pathogens. Infect Genet Evol 61:92–107. doi: 10.1016/j.meegid.2018.03.015 PubMed DOI

Matejková P, Strouhal M, Smajs D, Norris SJ, Palzkill T, Petrosino JF, Sodergren E, Norton JE, Singh J, Richmond TA, Molla MN, Albert TJ, Weinstock GM. 2008. Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays. BMC Microbiol 8:76. doi: 10.1186/1471-2180-8-76 PubMed DOI PMC

Giacani L, Jeffrey BM, Molini BJ, Le HT, Lukehart SA, Centurion-Lara A, Rockey DD. 2010. Complete genome sequence and annotation of the Treponema pallidum subsp. pallidum Chicago strain. J Bacteriol 192:2645–2646. doi: 10.1128/JB.00159-10 PubMed DOI PMC

Giacani L, Iverson-Cabral SL, King JCK, Molini BJ, Lukehart SA, Centurion-Lara A. 2014. Complete genome sequence of the Treponema pallidum subsp. pallidum Sea81-4 strain. Genome Announc 2:e00333-14. doi: 10.1128/genomeA.00333-14 PubMed DOI PMC

Pětrošová H, Zobaníková M, Čejková D, Mikalová L, Pospíšilová P, Strouhal M, Chen L, Qin X, Muzny DM, Weinstock GM, Šmajs D. 2012. Whole genome sequence of Treponema pallidum ssp. pallidum, strain Mexico A, suggests recombination between yaws and syphilis strains. PLoS Negl Trop Dis 6:e1832. doi: 10.1371/journal.pntd.0001832 PubMed DOI PMC

Zobaníková M, Mikolka P, Cejková D, Pospíšilová P, Chen L, Strouhal M, Qin X, Weinstock GM, Smajs D. 2012. Complete genome sequence of Treponema pallidum strain DAL-1. Stand Genomic Sci 7:12–21. doi: 10.4056/sigs.2615838 PubMed DOI PMC

Pětrošová H, Pospíšilová P, Strouhal M, Čejková D, Zobaníková M, Mikalová L, Sodergren E, Weinstock GM, Šmajs D. 2013. Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters. PLoS One 8:e74319. doi: 10.1371/journal.pone.0074319 PubMed DOI PMC

Hawley KL, Montezuma-Rusca JM, Delgado KN, Singh N, Uversky VN, Caimano MJ, Radolf JD, Luthra A. 2021. Structural modeling of the Treponema pallidum outer membrane protein repertoire: a road map for deconvolution of syphilis pathogenesis and development of a syphilis vaccine. J Bacteriol 203:e00082-21. doi: 10.1128/JB.00082-21 PubMed DOI PMC

Delgado KN, Montezuma-Rusca JM, Orbe IC, Caimano MJ, La Vake CJ, Luthra A, Hennelly CM, Nindo FN, Meyer JW, Jones LD, Parr JB, Salazar JC, Moody MA, Radolf JD, Hawley KL. 2022. Extracellular loops of the Treponema pallidum FadL orthologs TP0856 and TP0858 elicit IgG antibodies and IgG PubMed DOI PMC

Nechvátal L, Pětrošová H, Grillová L, Pospíšilová P, Mikalová L, Strnadel R, Kuklová I, Kojanová M, Kreidlová M, Vaňousová D, Procházka P, Zákoucká H, Krchňáková A, Smajs D. 2014. Syphilis-causing strains belong to separate SS14-like or nichols-like groups as defined by multilocus analysis of 19 Treponema pallidum strains. Int J Med Microbiol 304:645–653. doi: 10.1016/j.ijmm.2014.04.007 PubMed DOI

Grillova L, Jolley K, Šmajs D, Picardeau M. 2019. A public database for the new MLST scheme for Treponema pallidum subsp. pallidum: surveillance and epidemiology of the causative agent of syphilis. PeerJ 6:e6182. doi: 10.7717/peerj.6182 PubMed DOI PMC

Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842. doi: 10.1093/bioinformatics/btq033 PubMed DOI PMC

Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet j 17:10. doi: 10.14806/ej.17.1.200 DOI

Li H. 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094–3100. doi: 10.1093/bioinformatics/bty191 PubMed DOI PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup . 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079. doi: 10.1093/bioinformatics/btp352 PubMed DOI PMC

Breese MR, Liu Y. 2013. NGSUtils: a software suite for analyzing and manipulating next-generation sequencing datasets. Bioinformatics 29:494–496. doi: 10.1093/bioinformatics/bts731 PubMed DOI PMC

Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736. doi: 10.1101/gr.215087.116 PubMed DOI PMC

Vaser R, Sović I, Nagarajan N, Šikić M. 2017. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res 27:737–746. doi: 10.1101/gr.214270.116 PubMed DOI PMC

Murrell B, Weaver S, Smith MD, Wertheim JO, Murrell S, Aylward A, Eren K, Pollner T, Martin DP, Smith DM, Scheffler K, Kosakovsky Pond SL. 2015. Gene-wide identification of episodic selection. Mol Biol Evol 32:1365–1371. doi: 10.1093/molbev/msv035 PubMed DOI PMC

Wisotsky SR, Kosakovsky Pond SL, Shank SD, Muse SV. 2020. Synonymous site-to-site substitution rate variation dramatically inflates false positive rates of selection analyses: ignore at your own peril. Mol Biol Evol 37:2430–2439. doi: 10.1093/molbev/msaa037 PubMed DOI PMC

Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, et al. 2024. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630:493–500. doi: 10.1038/s41586-024-07487-w PubMed DOI PMC

Du Z, Su H, Wang W, Ye L, Wei H, Peng Z, Anishchenko I, Baker D, Yang J. 2021. The trRosetta server for fast and accurate protein structure prediction. Nat Protoc 16:5634–5651. doi: 10.1038/s41596-021-00628-9 PubMed DOI

Delgado KN, Caimano MJ, Orbe IC, Vicente CF, La Vake CJ, Grassmann AA, Moody MA, Radolf JD, Hawley KL. 2024. Immunodominant extracellular loops of Treponema pallidum FadL outer membrane proteins elicit antibodies with opsonic and growth-inhibitory activities. PLoS Pathog 20:e1012443. doi: 10.1371/journal.ppat.1012443 PubMed DOI PMC

Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega. Mol Syst Biol 7:539. doi: 10.1038/msb.2011.75 PubMed DOI PMC

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. 2004. UCSF chimera--a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. doi: 10.1002/jcc.20084 PubMed DOI

Desrosiers DC, Anand A, Luthra A, Dunham-Ems SM, LeDoyt M, Cummings MAD, Eshghi A, Cameron CE, Cruz AR, Salazar JC, Caimano MJ, Radolf JD. 2011. TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein. Mol Microbiol 80:1496–1515. doi: 10.1111/j.1365-2958.2011.07662.x PubMed DOI PMC

Doyle MT, Jimah JR, Dowdy T, Ohlemacher SI, Larion M, Hinshaw JE, Bernstein HD. 2022. Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding. Cell 185:1143–1156. doi: 10.1016/j.cell.2022.02.016 PubMed DOI PMC

Guest RL, Silhavy TJ. 2023. Cracking outer membrane biogenesis. Biochim Biophys Acta Mol Cell Res 1870:119405. doi: 10.1016/j.bbamcr.2022.119405 PubMed DOI PMC

Botte M, Ni D, Schenck S, Zimmermann I, Chami M, Bocquet N, Egloff P, Bucher D, Trabuco M, Cheng RKY, Brunner JD, Seeger MA, Stahlberg H, Hennig M. 2022. Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation. Nat Commun 13:1826. doi: 10.1038/s41467-022-29459-2 PubMed DOI PMC

Fraser CM, Norris SJ, Weinstock GM, White O, Sutton GG, Dodson R, Gwinn M, Hickey EK, Clayton R, Ketchum KA, et al. 1998. Complete genome sequence of Treponema pallidum , the syphilis spirochete . Science 281:375–388. doi: 10.1126/science.281.5375.375 PubMed DOI

Botos I, Majdalani N, Mayclin SJ, McCarthy JG, Lundquist K, Wojtowicz D, Barnard TJ, Gumbart JC, Buchanan SK. 2016. Structural and functional characterization of the LPS transporter LptDE from gram-negative pathogens. Structure 24:965–976. doi: 10.1016/j.str.2016.03.026 PubMed DOI PMC

Hearn EM, Patel DR, Lepore BW, Indic M, van den Berg B. 2009. Transmembrane passage of hydrophobic compounds through a protein channel wall. Nature 458:367–370. doi: 10.1038/nature07678 PubMed DOI PMC

Maděránková D, Mikalová L, Strouhal M, Vadják Š, Kuklová I, Pospíšilová P, Krbková L, Koščová P, Provazník I, Šmajs D. 2019. Identification of positively selected genes in human pathogenic treponemes: syphilis-, yaws-, and bejel-causing strains differ in sets of genes showing adaptive evolution. PLoS Negl Trop Dis 13:e0007463. doi: 10.1371/journal.pntd.0007463 PubMed DOI PMC

Pla-Díaz M, Sánchez-Busó L, Giacani L, Šmajs D, Bosshard PP, Bagheri HC, Schuenemann VJ, Nieselt K, Arora N, González-Candelas F. 2022. Evolutionary processes in the emergence and recent spread of the syphilis agent, Treponema pallidum. Mol Biol Evol 39:msab318. doi: 10.1093/molbev/msab318 PubMed DOI PMC

Strouhal M, Mikalová L, Haviernik J, Knauf S, Bruisten S, Noordhoek GT, Oppelt J, Čejková D, Šmajs D. 2018. Complete genome sequences of two strains of Treponema pallidum subsp. pertenue from Indonesia: modular structure of several treponemal genes. PLoS Negl Trop Dis 12:e0006867. doi: 10.1371/journal.pntd.0006867 PubMed DOI PMC

Brinkman MB, McGill MA, Pettersson J, Rogers A, Matejková P, Smajs D, Weinstock GM, Norris SJ, Palzkill T. 2008. A novel Treponema pallidum antigen, TP0136, is an outer membrane protein that binds human fibronectin. Infect Immun 76:1848–1857. doi: 10.1128/IAI.01424-07 PubMed DOI PMC

Cameron CE. 2003. Identification of a Treponema pallidum laminin-binding protein. Infect Immun 71:2525–2533. doi: 10.1128/IAI.71.5.2525-2533.2003 PubMed DOI PMC

Pospíšilová P, Grange PA, Grillová L, Mikalová L, Martinet P, Janier M, Vermersch A, Benhaddou N, Del Giudice P, Alcaraz I, Truchetet F, Dupin N, Šmajs D. 2018. Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: infecting treponemes are genetically diverse and belong to 18 allelic profiles. PLoS One 13:e0201068. doi: 10.1371/journal.pone.0201068 PubMed DOI PMC

Vrbová E, Pospíšilová P, Dastychová E, Kojanová M, Kreidlová M, Rob F, Vašků V, Mosio P, Strnadel R, Faustmannová O, Kuklová I, Heroldová MD, Zákoucká H, Šmajs D. 2024. Majority of Treponema pallidum ssp. pallidum MLST allelic profiles in the Czech Republic (2004-2022) belong to two SS14-like clusters. Sci Rep 14:17463. doi: 10.1038/s41598-024-68656-5 PubMed DOI PMC

Weinstock GM, Smajs D, Hardham J, Norris SJ. 2000. From microbial genome sequence to applications. Res Microbiol 151:151–158. doi: 10.1016/s0923-2508(00)00115-7 PubMed DOI

Cejková D, Zobaníková M, Chen L, Pospíšilová P, Strouhal M, Qin X, Mikalová L, Norris SJ, Muzny DM, Gibbs RA, Fulton LL, Sodergren E, Weinstock GM, Smajs D. 2012. Whole genome sequences of three Treponema pallidum ssp. pertenue strains: yaws and syphilis treponemes differ in less than 0.2% of the genome sequence. PLoS Negl Trop Dis 6:e1471. doi: 10.1371/journal.pntd.0001471 PubMed DOI PMC

Šmajs D, Norris SJ, Weinstock GM. 2012. Genetic diversity in Treponema pallidum: implications for pathogenesis, evolution and molecular diagnostics of syphilis and yaws. Infect Genet Evol 12:191–202. doi: 10.1016/j.meegid.2011.12.001 PubMed DOI PMC

Kumar S, Caimano MJ, Anand A, Dey A, Hawley KL, LeDoyt ME, La Vake CJ, Cruz AR, Ramirez LG, Paštěková L, Bezsonova I, Šmajs D, Salazar JC, Radolf JD. 2018. Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of Treponema pallidum subsp. pallidum, the syphilis spirochete. mBio 9:e01006-18. doi: 10.1128/mBio.01006-18 PubMed DOI PMC

Teelucksingh T, Thompson LK, Cox G. 2020. The evolutionary conservation of Escherichia coli drug efflux pumps supports physiological functions. J Bacteriol 202:e00367-20. doi: 10.1128/JB.00367-20 PubMed DOI PMC

Noda AA, Méndez M, Rodríguez I, Šmajs D. 2022. Genetic recombination in Treponema pallidum: implications for diagnosis epidemiology, and vaccine development. Sex Transm Dis 49. doi: 10.1097/OLQ.0000000000001497 PubMed DOI