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Although CD8\u25a1 T cells contribute to immune control of tuberculosis, the pathways through which bacterial antigens access major histocompatibility complex class I (MHC-I) antigen presentation remain incompletely defined. Here, we show that the activity of an Mtb secretion system actively promotes antigen presentation on MHC-I. Using quantitative immunopeptidomics, host and bacterial genetic perturbations, and T cell activation assays, we demonstrate that presentation of Mtb-derived peptides on MHC-I requires the ESX-1 type VII secretion system. Presentation of these peptides proceeds in a manner dependent on the transporter associated with antigen processing (TAP) but independent of host cell mechanisms such as autophagy or MPEG1-mediated pore formation. Chemical induction of phagosomal membrane damage fails to restore antigen presentation in the absence of ESX-1 activity, suggesting that pathogen-encoded secretion, not nonspecific membrane rupture, governs access to MHC-I antigen processing pathways. These findings reveal a secretion system\u2013driven mechanism of antigen presentation, redefining how mycobacteria interface with host MHC-I pathways, potentially informing tuberculosis vaccine design strategies, and highlighting a potential route for synthetic antigen delivery to the cytosol in therapeutics and vaccination.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>One-sentence summary<\/jats:title>\n                  <jats:p>\n                    Presentation of\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    antigens on MHC class I through a cytosolic pathway depends on a bacterial secretion system rather than host response and cross-presentation pathways.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.11.19.689332","type":"posted-content","created":{"date-parts":[[2025,11,20]],"date-time":"2025-11-20T17:30:13Z","timestamp":1763659813000},"source":"Crossref","is-referenced-by-count":1,"title":["Control of antigen presentation on MHC-I by a bacterial secretion system"],"prefix":"10.64898","author":[{"given":"Owen","family":"Leddy","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel","family":"McGinn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Allsup","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madison","family":"Leone","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Penny","family":"Timms","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matias","family":"Gutierrez-Gonzalez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmed S.","family":"Fahad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan","family":"Milligan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wilfredo F.","family":"Garcia-Beltran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brandon J.","family":"DeKosky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Forest M.","family":"White","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1716-6712","authenticated-orcid":false,"given":"Bryan D.","family":"Bryson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025112323200869000_2025.11.19.689332v1.1","doi-asserted-by":"crossref","unstructured":"Gonzales, G. 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