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This requires a safe <jats:italic>Mycobacterium tuberculosis<\/jats:italic> (Mtb) strain that can both replicate in the host and be reliably cleared. Here we genetically engineered Mtb strains encoding up to three kill switches: two mycobacteriophage lysin operons negatively regulated by tetracycline and a degron domain\u2013NadE fusion, which induces ClpC1-dependent degradation of the essential enzyme NadE, negatively regulated by trimethoprim. The triple-kill-switch (TKS) strain showed similar growth kinetics and antibiotic susceptibilities to wild-type Mtb under permissive conditions but was rapidly killed in vitro without trimethoprim and doxycycline. It established infection in mice receiving antibiotics but was rapidly cleared upon cessation of treatment, and no relapse was observed in infected severe combined immunodeficiency mice or Rag<jats:sup>\u2212\/\u2212<\/jats:sup> mice. The TKS strain had an escape mutation rate of less than 10<jats:sup>\u221210<\/jats:sup> per genome per generation. These findings suggest that the TKS strain could be a safe, effective candidate for a human challenge model.<\/jats:p>","DOI":"10.1038\/s41564-024-01913-5","type":"journal-article","created":{"date-parts":[[2025,1,10]],"date-time":"2025-01-10T10:02:54Z","timestamp":1736503374000},"page":"482-494","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Engineered Mycobacterium tuberculosis triple-kill-switch strain provides controlled tuberculosis infection in animal models"],"prefix":"10.1038","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6930-3347","authenticated-orcid":false,"given":"Xin","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongwei","family":"Su","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua B.","family":"Wallach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey C.","family":"Wagner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin J.","family":"Braunecker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Gardner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristine M.","family":"Guinn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole C.","family":"Howard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2765-1626","authenticated-orcid":false,"given":"Thais","family":"Klevorn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5181-4616","authenticated-orcid":false,"given":"Kan","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue J.","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4330-2389","authenticated-orcid":false,"given":"Yao","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Douaa","family":"Mugahid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9800-3362","authenticated-orcid":false,"given":"Mark","family":"Rodgers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaimie","family":"Sixsmith","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shoko","family":"Wakabayashi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junhao","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew","family":"Zimmerman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9470-5009","authenticated-orcid":false,"given":"V\u00e9ronique","family":"Dartois","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7874-8981","authenticated-orcid":false,"given":"JoAnne L.","family":"Flynn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philana Ling","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7951-2310","authenticated-orcid":false,"given":"Sabine","family":"Ehrt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7565-9975","authenticated-orcid":false,"given":"Sarah M.","family":"Fortune","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5120-962X","authenticated-orcid":false,"given":"Eric J.","family":"Rubin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4445-7219","authenticated-orcid":false,"given":"Dirk","family":"Schnappinger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,1,10]]},"reference":[{"key":"1913_CR1","unstructured":"Stop TB Partnership. 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