{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T16:08:44Z","timestamp":1790698124529,"version":"4.1.0"},"update-to":[{"DOI":"10.1371\/journal.ppat.1009612","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T00:00:00Z","timestamp":1626393600000}}],"reference-count":52,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2021,7,6]],"date-time":"2021-07-06T00:00:00Z","timestamp":1625529600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"national institutes of health","doi-asserted-by":"publisher","award":["U19AI144133"],"award-info":[{"award-number":["U19AI144133"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"national institutes of health","doi-asserted-by":"publisher","award":["U19AI144133"],"award-info":[{"award-number":["U19AI144133"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>\n                    Despite more than a century of research, genetic manipulation of\n                    <jats:italic>Treponema pallidum<\/jats:italic>\n                    subsp.\n                    <jats:italic>pallidum<\/jats:italic>\n                    (\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>pallidum<\/jats:italic>\n                    ), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>pallidum<\/jats:italic>\n                    success as a pathogen. A recently described method for\n                    <jats:italic>in vitro<\/jats:italic>\n                    cultivation of\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>pallidum<\/jats:italic>\n                    , however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the\n                    <jats:italic>tprA<\/jats:italic>\n                    (\n                    <jats:italic>tp0009<\/jats:italic>\n                    ) pseudogene in the SS14\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>pallidum<\/jats:italic>\n                    strain with a kanamycin resistance (\n                    <jats:italic>kan<\/jats:italic>\n                    <jats:sup>R<\/jats:sup>\n                    ) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the\n                    <jats:italic>kan<\/jats:italic>\n                    <jats:sup>R<\/jats:sup>\n                    gene was cloned downstream of the\n                    <jats:italic>tp0574<\/jats:italic>\n                    gene promoter. The\n                    <jats:italic>tp0574<\/jats:italic>\n                    prom-\n                    <jats:italic>kan<\/jats:italic>\n                    <jats:sup>R<\/jats:sup>\n                    cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the\n                    <jats:italic>tprA<\/jats:italic>\n                    pseudogene. To induce homologous recombination and integration of the\n                    <jats:italic>kan<\/jats:italic>\n                    <jats:sup>R<\/jats:sup>\n                    cassette into the\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>pallidum<\/jats:italic>\n                    chromosome,\n                    <jats:italic>in vitro<\/jats:italic>\n                    -cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl\n                    <jats:sub>2<\/jats:sub>\n                    -based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the\n                    <jats:italic>kan<\/jats:italic>\n                    <jats:sup>R<\/jats:sup>\n                    cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\/or\n                    <jats:italic>in vivo<\/jats:italic>\n                    . ddPCR analysis of RNA and mass spectrometry confirmed expression of the\n                    <jats:italic>kan<\/jats:italic>\n                    <jats:sup>R<\/jats:sup>\n                    message and protein in treponemes propagated\n                    <jats:italic>in vitro<\/jats:italic>\n                    . Moreover,\n                    <jats:italic>tprA<\/jats:italic>\n                    knockout (\n                    <jats:italic>tprA<\/jats:italic>\n                    <jats:sup>ko<\/jats:sup>\n                    -SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>pallidum<\/jats:italic>\n                    is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1009612","type":"journal-article","created":{"date-parts":[[2021,7,6]],"date-time":"2021-07-06T13:42:53Z","timestamp":1625578973000},"page":"e1009612","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":61,"title":["Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7295-9531","authenticated-orcid":true,"given":"Emily","family":"Romeis","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5788-0465","authenticated-orcid":true,"given":"Lauren","family":"Tantalo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9334-8150","authenticated-orcid":true,"given":"Nicole","family":"Lieberman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5499-3392","authenticated-orcid":true,"given":"Quynh","family":"Phung","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex","family":"Greninger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3889-7274","authenticated-orcid":true,"given":"Lorenzo","family":"Giacani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,7,6]]},"reference":[{"key":"ppat.1009612.ref001","volume-title":"Prevalence 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