Treponema pallidum subsp. pallidum (T. pallidum), the fastidious causative agent of syphilis, has become more accessible for research with its recently developed in vitro cultivation method. In this work, the proteomes of seven T. pallidum strains (Nichols-like: DAL-1, Haiti B, and Madras; SS14-like: SS14, Mexico A, Philadelphia 1, and Grady), cultivated in vitro, were analyzed in biological triplicates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS data were processed against their corresponding genomes using various annotation algorithms (DFAST, PGAP, Prodigal, Prokka, RAST, GeneMarkS, and manual GenBank annotation). Additionally, ORFfinder was used to predict all ORFs encoding polypeptides exceeding 50 amino acids. While the RAST algorithm predicted the highest number of genes per genome, GeneMarkS offered the best coverage of annotated genes (up to 88.9%). By combining annotations from seven T. pallidum strains, we identified 911 unique treponemal proteins (74.9% of 1216 predicted sequences). The confidence of protein identifications was high, with 85.5% identified by two or more peptides and 72.4% by three or more peptides. Overall, 51 proteins showed statistically significant quantitative differences in intensity across T. pallidum strains. Furthermore, our proteome analysis revealed detectable quantitative proteomic differences between strains in the Nichols-like and SS14-like groups.

Department of Biology Faculty of Medicine Masaryk University Brno 625 00 Czech Republic

National Centre for Biomolecular Research Central European Institute of Technology and Faculty of Science Masaryk University Brno 625 00 Czech Republic

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Newman L., Rowley J., Vander Hoorn S., Wijesooriya N. S., Unemo M., Low N., Stevens G., Gottlieb S., Kiarie J., Temmerman M.. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One. 2015;10(12):e0143304. doi: 10.1371/journal.pone.0143304. PubMed DOI PMC

Peeling R., Mabey D., Kamb M., Chen X., Radolf J. D., Benzaken A.. Syphilis. Nat. Rev. Dis. Prim. 2017;3:e17073. doi: 10.1038/nrdp.2017.73. PubMed DOI PMC

McGill M. A., Edmondson D. G., Carroll J. A., Cook R. G., Orkiszewski R. S., Norris S. J.. Characterization and serologic analysis of the Treponema pallidum proteome. Infect. Immun. 2010;78(6):2631–2643. doi: 10.1128/IAI.00173-10. PubMed DOI PMC

Osbak K. K., Houston S., Lithgow K. V., Meehan C. J., Strouhal M., Šmajs D., Cameron C. E., Van Ostade X., Kenyon C. R., Van Raemdonck G. A.. Characterizing the syphilis-causing Treponema pallidum ssp. pallidum proteome using complementary mass spectrometry. PLoS Negl. Trop. Dis. 2016;10(9):e0004988. doi: 10.1371/journal.pntd.0004988. PubMed DOI PMC

Houston S., Gomez A., Geppert A., Eshghi A., Smith D. S., Waugh S., Hardie D. B., Goodlett D. R., Cameron C. E.. Deep proteome coverage advances knowledge of Treponema pallidum protein expression profiles during infection. Sci. Rep. 2023;13(1):e18259. doi: 10.1038/s41598-023-45219-8. PubMed DOI PMC

Houston S., Gomez A., Geppert A., Goodyear M. C., Cameron C. E.. In-depth proteome coverage of in vitro-cultured Treponema pallidum and quantitative comparison analyses with in vivo-grown treponemes. J. Proteome Res. 2024;23(5):1725–1743. doi: 10.1021/acs.jproteome.3c00891. PubMed DOI PMC

Houston S., Marshall S., Gomez A., Cameron C. E.. Proteomic analysis of the Treponema pallidum subsp. pallidum SS14 strain: Coverage and comparison with the Nichols strain proteome. Front. Microbiol. 2024;15:e1505893. doi: 10.3389/fmicb.2024.1505893. PubMed DOI PMC

Edmondson D. G., Hu B., Norris S. J.. Long-term in vitro culture of the syphilis spirochete Treponema pallidum subsp. pallidum . MBio. 2018;9(3):e01153-18. doi: 10.1128/mBio.01153-18. 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., Šmajs D.. 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. 2014;304(5–6):645–653. doi: 10.1016/j.ijmm.2014.04.007. PubMed DOI

Pětrošová H., Pospíšilová P., Strouhal M., Čejková D., Zobaníková M., Mikalová L., Sodergren E., Weinstock G. M., Šmajs D.. Resequencing of Treponema pallidum ssp. pallidum strains Nichols and SS14: Correction of sequencing errors resulted in increased separation of syphilis treponeme subclusters. PLoS One. 2013;8(9):e74319. doi: 10.1371/journal.pone.0074319. PubMed DOI PMC

Arora N., Schuenemann V. J., Jäger G., Peltzer A., Seitz A., Herbig A., Strouhal M., Grillová L., Sánchez-Busó L., Kühnert D., Bos K. I., Davis L. R., Mikalová L., Bruisten S., Komericki P., French P., Grant P. R., Pando M. A., Vaulet L. G., Fermepin M. R., Martinez A., Centurion Lara A., Giacani L., Norris S. J., Šmajs D., Bosshard P. P., González-Candelas F., Nieselt K., Krause J., Bagheri H. C.. Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster. Nat. Microbiol. 2017;2:e16245. doi: 10.1038/nmicrobiol.2016.245. PubMed DOI

Šmajs D., Mikalová L., Strouhal M., Grillová L.. Why there are two genetically distinct syphilis-causing strains? Forum Immun Dis Ther. 2016;7(3–4):181–190. doi: 10.1615/ForumImmunDisTher.2017020184. DOI

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

Bosák J., Mikalová L., Hrala M., Pospíšilová P., Faldyna M., Šmajs D.. Treponema pallidum subsp. pallidum strains DAL-1 and Philadelphia 1 differ in generation times in vitro as well as during experimental rabbit infection. PLoS One. 2024;19(5):e0304033. doi: 10.1371/journal.pone.0304033. PubMed DOI PMC

Edmondson D. G., Norris S. J.. In vitro cultivation of the syphilis spirochete Treponema pallidum . Curr. Protoc. 2021;1(2):e44. doi: 10.1002/cpz1.44. PubMed DOI PMC

Wiśniewski J. R., Ostasiewicz P., Mann M.. High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers. J. Proteome Res. 2011;10(7):3040–3049. doi: 10.1021/pr200019m. PubMed DOI

Yeung Y. G., Nieves E., Angeletti R. H., Stanley E. R.. Removal of detergents from protein digests for Mass spectrometry analysis. Anal. Biochem. 2008;382(2):135–137. doi: 10.1016/j.ab.2008.07.034. PubMed DOI PMC

Martinková P., Konečná H., Gintar P., Kryštofová K., Potěšil D., Trtílek M., Zdráhal Z.. Benchmarking of two peptide clean-up protocols: SP2 and ethyl acetate extraction for sodium dodecyl sulfate or polyethylene glycol removal from plant samples before LC-MS/MS. Int. J. Mol. Sci. 2023;24(24):No. e17347. doi: 10.3390/ijms242417347. PubMed DOI PMC

Demichev V., Messner C. B., Vernardis S. I., Lilley K. S., Ralser M.. DIA-NN: Neural networks and interference correction enable deep proteome coverage in high throughput. Nat. Methods. 2020;17(1):41–44. doi: 10.1038/s41592-019-0638-x. PubMed DOI PMC

Perez-Riverol Y., Bandla C., Kundu D. J., Kamatchinathan S., Bai J., Hewapathirana S., John N. S., Prakash A., Walzer M., Wang S., Vizcaíno J. A.. The PRIDE database at 20 years: 2025 Update. Nucleic Acids Res. 2025;53(D1):D543–D553. doi: 10.1093/nar/gkae1011. PubMed DOI PMC

Tanizawa Y., Fujisawa T., Nakamura Y.. DFAST: A flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics. 2018;34(6):1037–1039. doi: 10.1093/bioinformatics/btx713. PubMed DOI PMC

Li W., O’Neill K. R., Haft D. H., Dicuccio M., Chetvernin V., Badretdin A., Coulouris G., Chitsaz F., Derbyshire M. K., Durkin A. S., Gonzales N. R., Gwadz M., Lanczycki C. J., Song J. S., Thanki N., Wang J., Yamashita R. A., Yang M., Zheng C., Marchler-Bauer A., Thibaud-Nissen F.. RefSeq: Expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Res. 2021;49(D1):D1020–D1028. doi: 10.1093/nar/gkaa1105. PubMed DOI PMC

Hyatt D., Chen G. L., LoCascio P. F., Land M. L., Larimer F. W., Hauser L. J.. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:e119. doi: 10.1186/1471-2105-11-119. PubMed DOI PMC

Seemann T.. Prokka: Rapid Prokaryotic Genome Annotation. Bioinformatics. 2014;30(14):2068–2069. doi: 10.1093/bioinformatics/btu153. PubMed DOI

Brettin T., Davis J. J., Disz T., Edwards R. A., Gerdes S., Olsen G. J., Olson R., Overbeek R., Parrello B., Pusch G. D., Shukla M., Thomason J. A., Stevens R., Vonstein V., Wattam A. R., Xia F.. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci. Rep. 2015;5:e8365. doi: 10.1038/srep08365. PubMed DOI PMC

Lomsadze A., Gemayel K., Tang S., Borodovsky M.. Modeling Leaderless Transcription and atypical genes results in more accurate gene prediction in prokaryotes. Genome Res. 2018;28(7):1079–1089. doi: 10.1101/gr.230615.117. PubMed DOI PMC

Wheeler D. L., Church D. M., Edgar R., Federhen S., Helmberg W., Madden T. L., Pontius J. U., Schuler G. D., Schriml L. M., Sequeira E., Suzek T. O., Tatusova T. A., Wagner L.. Database resources of the National Center for Biotechnology Information: Update. Nucleic Acids Res. 2004;32(suppl_1):D35–D40. doi: 10.1093/NAR/GKH073. PubMed DOI PMC

Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T. L.. BLAST+: Architecture and applications. BMC Bioinformatics. 2009;10:e421. doi: 10.1186/1471-2105-10-421. PubMed DOI PMC

Fraser C. M., Norris S. J., Weinstock G. M., White O., Sutton G. G., Dodson R., Gwinn M., Hickey E. K., Clayton R., Ketchum K. A., Sodergren E., Hardham J. M., McLeod M. P., Salzberg S., Peterson J., Khalak H., Richardson D., Howell J. K., Chidambaram M., Utterback T., McDonald L., Artiach P., Bowman C., Cotton M. D., Fujii C., Garland S., Hatch B., Horst K., Roberts K., Sandusky M., Weidman J., Smith H. O., Venter J. C.. Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science (80-.). 1998;281(5375):375–388. doi: 10.1126/science.281.5375.375. PubMed DOI

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

Grillová L., Oppelt J., Mikalová L., Nováková M., Giacani L., Niesnerová A., Noda A. A., Mechaly A. E., Pospíšilová P., Čejková D., Grange P. A., Dupin N., Strnadel R., Chen M., Denham I., Arora N., Picardeau M., Weston C., Forsyth R. A., Šmajs D.. Directly sequenced genomes of contemporary strains of syphilis reveal recombination-driven diversity in genes encoding predicted surface-exposed antigens. Front. Microbiol. 2019;10:e1691. doi: 10.3389/fmicb.2019.01691. PubMed DOI PMC

Šmajs D., McKevitt M., Howell J. K., Norris S. J., Cai W. W., Palzkill T., Weinstock G. M.. Transcriptome of Treponema pallidum: Gene expression profile during experimental rabbit infection. J. Bacteriol. 2005;187(5):1866–1874. doi: 10.1128/JB.187.5.1866-1874.2005. PubMed DOI PMC

de Lay B. D., Cameron T. A., de Lay N. R., Norris S. J., Edmondson D. G.. Comparison of transcriptional profiles of Treponema pallidum during experimental infection of rabbits and in vitro culture: Highly similar, yet different. PLoS Pathog. 2021;17(9):e1009949. doi: 10.1371/journal.ppat.1009949. PubMed DOI PMC

Čejková D., Zobaníková M., Chen L., Pospíšilová P., Strouhal M., Qin X., Mikalová L., Norris S. J., Muzny D. M., Gibbs R. A., Fulton L. L., Sodergren E., Weinstock G. M., Šmajs D.. 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. 2012;6(1):e1471. doi: 10.1371/journal.pntd.0001471. PubMed DOI PMC

Edmondson D. G., Delay B. D., Kowis L. E., Norris S. J.. Parameters affecting continuous in vitro culture of Treponema pallidum strains. MBio. 2021;12(1):1–21. doi: 10.1128/mBio.03536-20. PubMed DOI PMC

Taouk M. L., Taiaroa G., Pasricha S., Herman S., Chow E. P. F., Azzatto F., Zhang B., Sia C. M., Duchene S., Lee A., Higgins N., Prestedge J., Lee Y. W., Thomson N. R., Graves B., Meumann E., Gunathilake M., Hocking J. S., Bradshaw C. S., Beale M. A., Howden B. P., Chen M. Y., Fairley C. K., Ingle D. J., Williamson D. A.. Characterisation of Treponema pallidum lineages within the contemporary syphilis outbreak in Australia: A genomic epidemiological analysis. Lancet. Microbe. 2022;3(6):417–426. doi: 10.1016/S2666-5247(22)00035-0. PubMed DOI

Pospíšilová P., Grange P. A., Grillová L., Mikalová L., Martinet P., Janier M., Vermersch A., Benhaddou N., Del Giudice P., Alcaraz I., Truchetet F., Dupin N., Šmajs D.. 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. 2018;13(7):e0201068. doi: 10.1371/journal.pone.0201068. PubMed DOI PMC