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The semi-invariant antigen recognition receptor of MAIT cells specifically detects the non-polymorphic antigen-presenting molecule, major histocompatibility complex class I-related protein 1 (MR1), which primarily binds riboflavin-derived metabolites of microbial origin. To further interrogate the dependence of these antigens on riboflavin biosynthesis in mycobacteria, we deleted individual genes in the riboflavin biosynthesis pathways in\n                    <jats:italic>Mycobacterium smegmatis<\/jats:italic>\n                    (Msm) and\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    (Mtb) and evaluated the impact thereof on MAIT cell activation. Blocking the early steps of the pathway by deletion of RibA2 or RibG profoundly reduced, but did not completely ablate, MAIT cell activation by Msm or Mtb, whereas deletion of RibC, which catalyzes the last step in the pathway, had no significant effect. Interestingly, deletion of the lumazine synthase (RibH) specifically enhanced MAIT cell recognition of Mtb whereas loss of lumazine synthase activity had no impact on MAIT cell activation by Msm. MAIT cell activation by Msm was likewise unaffected by blocking the production of the MAIT cell antagonist, F\n                    <jats:sub>o<\/jats:sub>\n                    (by inhibiting its conversion from the riboflavin pathway intermediate, 5-amino-6-D-ribitylaminouracil (5-A-RU), through the deletion of\n                    <jats:italic>fbiC<\/jats:italic>\n                    ). Together, these results confirm a central role for 5-A-RU in generating mycobacterial MR1 ligands and reveal similarities and differences between Msm and Mtb in terms of the impact of riboflavin pathway disruption on MAIT cell activation.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1012632","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T14:02:50Z","timestamp":1751378570000},"page":"e1012632","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":15,"title":["Disruption of riboflavin biosynthesis in mycobacteria establishes riboflavin pathway intermediates as key precursors of MAIT cell agonists"],"prefix":"10.1371","volume":"21","author":[{"given":"Melissa D.","family":"Chengalroyen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nurudeen","family":"Oketade","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aneta","family":"Worley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megan","family":"Lucas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luisa Maria Nieto","family":"Ramirez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mabule L.","family":"Raphela","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gwendolyn M.","family":"Swarbrick","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul 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