{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T06:04:03Z","timestamp":1782281043669,"version":"3.54.5"},"posted":{"date-parts":[[2026,2,2]]},"group-title":"Microbiology","reference-count":41,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,2]],"date-time":"2026-02-02T00:00:00Z","timestamp":1769990400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH\/NIAID","award":["AI188782"],"award-info":[{"award-number":["AI188782"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Mycobacteria have a hydrophobic cell envelope that makes uniform growth in liquid culture challenging. Non-ionic detergents including Tween-80 and tyloxapol are commonly added to media when culturing mycobacterial species in the laboratory. Tyloxapol was reported to exhibit anti-tuberculous activity during animal infection with\n                  <jats:italic>M. tuberculosis<\/jats:italic>\n                  in the 1950s. In the 1980s, microscopy studies suggested that tyloxapol impacted the inter-action between\n                  <jats:italic>M. tuberculosis<\/jats:italic>\n                  and the phagosomal membrane, preventing mycobacterial access to the cytoplasm. It is now known that the ESX-1 Type VII secretion system mediates the interaction between pathogenic mycobacteria and the phagosomal membrane.\n                  <jats:italic>Mycobacterium marinum<\/jats:italic>\n                  is a pathogenic mycobacterial species that has been widely used to understand the molecular mechanisms and host responses to the ESX-1 system. The hemolytic activity of\n                  <jats:italic>M. marinum<\/jats:italic>\n                  allows the study of ESX-1 lytic activity outside of the context of a host cell. We found that tyloxapol inhibits the hemolytic activity of\n                  <jats:italic>M. marinum<\/jats:italic>\n                  in a concentration dependent manner. The addition of 100-fold less tyloxapol than commonly used for mycobacterial growth differentially inhibits the production and secretion of ESX-1 substrates required for lytic activity. Our findings directly impact how the field interprets data from studies where\n                  <jats:italic>M. marinum<\/jats:italic>\n                  , and potentially other mycobacterial species were grown in tyloxapol. Our findings may explain the original ob-servations linking tyloxapol to anti-tuberculosis activity.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary<\/jats:title>\n                  <jats:p>\n                    Tuberculosis, which is caused by\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    , is one of the world\u2019s deadliest diseases. We lack a clear understanding of how\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    and related mycobacterial species cause disease. In the 1950\u2019s, it was reported that treating\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    infected animals with tyloxapol improved the survival and in some cases protected the animals from death. Tyloxapol is a detergent that is commonly added to mycobacterial cultures to promote dispersed growth in the laboratory. Later studies suggested that tyloxapol altered the interaction between\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    and the phagosomal membrane during macrophage infection. The ability to escape the phagosome is essential for mycobacteria to cause disease, and is mediated by a Type VII protein secretion system, ESX-1. Using\n                    <jats:italic>M. marinum<\/jats:italic>\n                    , a well-established model for understanding the molecular mechanisms of ESX-1 secretion, we show that tyloxapol used at more than 100-fold less than what is commonly used to grow mycobacteria in the lab, inhibits ESX-1 secretion. Our findings have widespread implications on how we interpret our findings as a field, and may explain why tyloxapol impacted\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    infection of both animals and macrophages. Our study also indicates that tyloxapol can be used as a tool to understand the molecular mechanisms of ESX-1 protein secretion.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.02.02.703260","type":"posted-content","created":{"date-parts":[[2026,2,3]],"date-time":"2026-02-03T03:35:14Z","timestamp":1770089714000},"source":"Crossref","is-referenced-by-count":1,"title":["Tyloxapol inhibits ESX-1 secretion in\n                  <i>Mycobacterium marinum<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-1736-5770","authenticated-orcid":false,"given":"Owen A.","family":"Collars","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, University of Notre Dame; Notre Dame, 46556, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6733-596X","authenticated-orcid":false,"given":"Patricia A.","family":"Champion","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Notre Dame; Notre Dame, 46556, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026020511525168000_2026.02.02.703260v1.1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.86.5.1057-1069.1963"},{"key":"2026020511525168000_2026.02.02.703260v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.coi.2004.05.011"},{"key":"2026020511525168000_2026.02.02.703260v1.3","doi-asserted-by":"crossref","unstructured":"Chirakos AE , Balaram A , Conrad W , Champion PA . 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