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In this study, we leveraged a library of conditional mutants targeting 467 essential Mtb genes to characterize the chemical\u2013genetic interactions (CGIs) with TB drugs directly in the mouse infection model. We found that these in vivo CGIs differ significantly from those identified in vitro. Both drug-specific and drug-agnostic effects were identified, and many were preserved during treatment with a multidrug combination, suggesting numerous strategies for enhancing therapy. This work also elucidated the complex effects of pyrazinamide (PZA), a drug that relies on aspects of the infection environment for efficacy. Specifically, our work supports the importance of coenzyme A synthesis- inhibition during infection, as well as the antagonistic effect of iron limitation on PZA activity. In addition, we found that inhibition of thiamine and purine synthesis increases PZA efficacy, suggesting additional therapeutically exploitable metabolic dependencies. Our findings present a map of the unique in vivo CGIs, characterizing the mechanism of PZA activity in vivo and identifying potential targets for TB drug development.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2417525122","type":"journal-article","created":{"date-parts":[[2025,2,24]],"date-time":"2025-02-24T15:08:51Z","timestamp":1740409731000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":6,"title":["Chemical genetic interactions elucidate pathways controlling tuberculosis antibiotic efficacy during infection"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7451-4993","authenticated-orcid":false,"given":"Peter O.","family":"Oluoch","sequence":"first","affiliation":[{"name":"Department of Microbiology, University of Massachusetts Medical School","place":["Worcester"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2220-5229","authenticated-orcid":false,"given":"Eun-Ik","family":"Koh","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts Medical School","place":["Worcester"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9524-8302","authenticated-orcid":false,"given":"Megan K.","family":"Proulx","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts Medical School","place":["Worcester"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5579-5881","authenticated-orcid":false,"given":"Charlotte J.","family":"Reames","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts Medical School","place":["Worcester"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7837-1344","authenticated-orcid":false,"given":"Kadamba G.","family":"Papavinasasundaram","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts Medical School","place":["Worcester"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3677-2876","authenticated-orcid":false,"given":"Kenan C.","family":"Murphy","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts Medical School","place":["Worcester"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew D.","family":"Zimmerman","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation, Hackensack Meridian Health","place":["Nutley"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9470-5009","authenticated-orcid":false,"given":"V\u00e9ronique","family":"Dartois","sequence":"additional","affiliation":[{"name":"Center for Discovery and Innovation, Hackensack Meridian Health","place":["Nutley"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6178-4329","authenticated-orcid":false,"given":"Christopher M.","family":"Sassetti","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts Medical School","place":["Worcester"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,2,24]]},"reference":[{"key":"e_1_3_4_1_2","volume-title":"Global Tuberculosis Report 2023","author":"World Health Organization","year":"2023","unstructured":"World Health Organization, Global Tuberculosis Report 2023 (World Health Organization, 2023)."},{"key":"e_1_3_4_2_2","first-page":"325","article-title":"Short-course chemotherapy for pulmonary tuberculosis","volume":"111","author":"Fox W.","year":"1975","unstructured":"W. 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