{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T06:03:18Z","timestamp":1782280998257,"version":"3.54.5"},"posted":{"date-parts":[[2025,10,24]]},"group-title":"Microbiology","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,24]],"date-time":"2025-10-24T00:00:00Z","timestamp":1761264000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"NIH","award":["R01AI175972"],"award-info":[{"award-number":["R01AI175972"]}]},{"name":"NIH","award":["R01AI173328"],"award-info":[{"award-number":["R01AI173328"]}]},{"name":"NIH","award":["R01AI175972"],"award-info":[{"award-number":["R01AI175972"]}]},{"name":"NIH","award":["R21AI159348"],"award-info":[{"award-number":["R21AI159348"]}]},{"name":"NIH","award":["T32AI070117"],"award-info":[{"award-number":["T32AI070117"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,24]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Antibiotics targeting the mycobacterial cell wall are a cornerstone of tuberculosis treatment. Understanding how these drugs facilitate bacterial killing has important implications for identifying novel therapeutic targets and optimizing drug combinations. Previous studies on mycobacteria using the BacTiter-Glo\u2122 luminescence assay have reported increased ATP levels after treatment with cell wall inhibitors such as isoniazid, a phenomenon referred to as an \u2018ATP burst\u2019. This phenomenon has been proposed to contribute to drug-induced killing. In this study, we demonstrate that the ATP burst is not a biological response but rather an experimental artifact resulting from enhanced cell lysis induced by cell wall-targeting drugs. The addition of a bead-beating lysis step prior to luminescence measurement abolishes the ATP burst, enabling a more reliable assessment of intracellular ATP levels with this assay. Our findings thus establish a direct link between the ATP burst and the canonical mechanism of action of cell wall inhibitors. We further demonstrate the utility of this approach as a functional readout for identifying compounds that disrupt the mycobacterial cell wall and for screening synergistic or antagonistic interactions with known cell wall-targeting agents. Together, our results elucidate the mechanistic basis of the ATP burst and provide a valuable tool for antimycobacterial drug discovery and development.<\/jats:p>","DOI":"10.1101\/2025.10.24.684413","type":"posted-content","created":{"date-parts":[[2025,10,25]],"date-time":"2025-10-25T06:25:24Z","timestamp":1761373524000},"source":"Crossref","is-referenced-by-count":1,"title":["The mycobacterial ATP burst is a lysis artifact and serves as an assay for drug-induced cell wall damage"],"prefix":"10.64898","author":[{"given":"Claire V.","family":"Mulholland","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bei Shi","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Chong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinhua","family":"Cui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Pethe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Berney","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025102805350490000_2025.10.24.684413v1.1","unstructured":"WHO (2024). 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