{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T13:09:55Z","timestamp":1778332195138,"version":"3.51.4"},"posted":{"date-parts":[[2025,11,2]]},"group-title":"Microbiology","reference-count":118,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,2]],"date-time":"2025-11-02T00:00:00Z","timestamp":1762041600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000057","name":"NIGMS","doi-asserted-by":"crossref","award":["1R01AI178975-01"],"award-info":[{"award-number":["1R01AI178975-01"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant\n                  <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                  strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacterium\u2019s complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumulation in live mycobacterial cells. Here, we present a split-luciferin-based assay to quantify molecular accumulation in mycobacteria. Using this approach, we quantified the cytosolic accumulation of diverse small-molecule antibiotics and polyarginine peptides conjugated via a disulfide-linked D-cysteine tag. We also show the localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, demonstrating that these peptides can cross multiple accumulation barriers. Our findings establish the first assay for real-time quantification of cytosolic molecular accumulation in live mycobacteria, addressing a longstanding methodological gap and enabling mechanistic insights into intracellular drug uptake.\n                <\/jats:p>","DOI":"10.1101\/2025.11.02.686125","type":"posted-content","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T03:55:11Z","timestamp":1762142111000},"source":"Crossref","is-referenced-by-count":0,"title":["Split-luciferin Assay for Real-time Measurement of Cytosolic Molecular Accumulation in Live Mycobacteria"],"prefix":"10.64898","author":[{"given":"Rachita","family":"Dash","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrew","family":"Spira","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Irene","family":"Lepori","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah E.","family":"Newkirk","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sobika","family":"Bhandari","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noah","family":"Lucas","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mahendra D.","family":"Chordia","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M. Sloan","family":"Siegrist","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Massachusetts"},{"name":"Molecular and Cellular Biology Graduate Program, University of Massachusetts"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5676-0725","authenticated-orcid":false,"given":"Marcos M.","family":"Pires","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Virginia"},{"name":"Department of Microbiology, Immunology, and Cancer, University of Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026050905200795000_2025.11.02.686125v3.1","unstructured":"World Health Organization. Global Tuberculosis Report 2024. https:\/\/www.who.int\/teams\/global-programme-on-tuberculosis-and-lung-health\/tb-reports\/global-tuberculosis-report-2024\/tb-disease-burden."},{"key":"2026050905200795000_2025.11.02.686125v3.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-024-01025-1"},{"key":"2026050905200795000_2025.11.02.686125v3.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-019-0273-7"},{"key":"2026050905200795000_2025.11.02.686125v3.4","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1403078111"},{"key":"2026050905200795000_2025.11.02.686125v3.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-12718-4"},{"key":"2026050905200795000_2025.11.02.686125v3.6","doi-asserted-by":"publisher","DOI":"10.1021\/mp4000822"},{"key":"2026050905200795000_2025.11.02.686125v3.7","doi-asserted-by":"publisher","DOI":"10.1038\/nrd2199"},{"key":"2026050905200795000_2025.11.02.686125v3.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1310333110"},{"key":"2026050905200795000_2025.11.02.686125v3.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2014.07.012S0003-2697(14)00306-6"},{"key":"2026050905200795000_2025.11.02.686125v3.10","doi-asserted-by":"publisher","DOI":"10.1021\/cb5003015"},{"key":"2026050905200795000_2025.11.02.686125v3.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2017.01.076"},{"key":"2026050905200795000_2025.11.02.686125v3.12","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0038624"},{"key":"2026050905200795000_2025.11.02.686125v3.13","doi-asserted-by":"publisher","DOI":"10.1021\/ac504880r"},{"key":"2026050905200795000_2025.11.02.686125v3.14","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.5b08960"},{"key":"2026050905200795000_2025.11.02.686125v3.15","doi-asserted-by":"publisher","DOI":"10.1038\/srep17968"},{"key":"2026050905200795000_2025.11.02.686125v3.16","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202313870"},{"key":"2026050905200795000_2025.11.02.686125v3.17","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.2c00435"},{"key":"2026050905200795000_2025.11.02.686125v3.18","doi-asserted-by":"publisher","DOI":"10.1101\/2023.06.20.545103"},{"key":"2026050905200795000_2025.11.02.686125v3.19","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.3c00684"},{"key":"2026050905200795000_2025.11.02.686125v3.20","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.4c01062"},{"key":"2026050905200795000_2025.11.02.686125v3.21","doi-asserted-by":"publisher","DOI":"10.1101\/2025.02.27.640664"},{"key":"2026050905200795000_2025.11.02.686125v3.22","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.5c00330"},{"key":"2026050905200795000_2025.11.02.686125v3.23","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202217777"},{"key":"2026050905200795000_2025.11.02.686125v3.24","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-021-00598-y"},{"key":"2026050905200795000_2025.11.02.686125v3.25","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.8b00083"},{"key":"2026050905200795000_2025.11.02.686125v3.26","doi-asserted-by":"publisher","DOI":"10.1021\/cb5003015"},{"key":"2026050905200795000_2025.11.02.686125v3.27","doi-asserted-by":"publisher","DOI":"10.1101\/2024.12.15.628588"},{"key":"2026050905200795000_2025.11.02.686125v3.28","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.9b00439"},{"key":"2026050905200795000_2025.11.02.686125v3.29","doi-asserted-by":"publisher","DOI":"10.1016\/j.drudis.2018.05.022"},{"key":"2026050905200795000_2025.11.02.686125v3.30","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00337-22"},{"key":"2026050905200795000_2025.11.02.686125v3.31","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0010777"},{"key":"2026050905200795000_2025.11.02.686125v3.32","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbagen.2008.01.008"},{"key":"2026050905200795000_2025.11.02.686125v3.33","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.00008-08"},{"key":"2026050905200795000_2025.11.02.686125v3.34","doi-asserted-by":"publisher","DOI":"10.1007\/s00203-002-0469-4"},{"key":"2026050905200795000_2025.11.02.686125v3.35","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2017.7074"},{"key":"2026050905200795000_2025.11.02.686125v3.36","doi-asserted-by":"publisher","DOI":"10.1021\/ja309078t"},{"key":"2026050905200795000_2025.11.02.686125v3.37","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.1c00093"},{"key":"2026050905200795000_2025.11.02.686125v3.38","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.9b00439"},{"key":"2026050905200795000_2025.11.02.686125v3.39","doi-asserted-by":"publisher","DOI":"10.1016\/j.febslet.2006.08.073"},{"key":"2026050905200795000_2025.11.02.686125v3.40","doi-asserted-by":"publisher","DOI":"10.1016\/j.tetlet.2005.12.033"},{"key":"2026050905200795000_2025.11.02.686125v3.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2009.09.014"},{"key":"2026050905200795000_2025.11.02.686125v3.42","doi-asserted-by":"publisher","DOI":"10.1039\/C8RA02312C"},{"key":"2026050905200795000_2025.11.02.686125v3.43","doi-asserted-by":"publisher","DOI":"10.1021\/acs.bioconjchem.3c00381"},{"key":"2026050905200795000_2025.11.02.686125v3.44","doi-asserted-by":"publisher","DOI":"10.1111\/php.13814"},{"key":"2026050905200795000_2025.11.02.686125v3.45","doi-asserted-by":"publisher","DOI":"10.1039\/D0CS01492C"},{"key":"2026050905200795000_2025.11.02.686125v3.46","doi-asserted-by":"publisher","DOI":"10.1039\/D4CB00148F"},{"key":"2026050905200795000_2025.11.02.686125v3.47","doi-asserted-by":"publisher","DOI":"10.1021\/ja104525m"},{"key":"2026050905200795000_2025.11.02.686125v3.48","doi-asserted-by":"publisher","DOI":"10.1002\/9780470559277.ch140047"},{"key":"2026050905200795000_2025.11.02.686125v3.49","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0259181"},{"key":"2026050905200795000_2025.11.02.686125v3.50","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-11-35"},{"key":"2026050905200795000_2025.11.02.686125v3.51","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01013-12"},{"key":"2026050905200795000_2025.11.02.686125v3.52","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02428-14"},{"key":"2026050905200795000_2025.11.02.686125v3.53","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.00494"},{"key":"2026050905200795000_2025.11.02.686125v3.54","doi-asserted-by":"publisher","DOI":"10.1002\/ddr.21468"},{"key":"2026050905200795000_2025.11.02.686125v3.55","doi-asserted-by":"publisher","DOI":"10.1038\/nrd2468"},{"key":"2026050905200795000_2025.11.02.686125v3.56","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejps.2021.105831"},{"key":"2026050905200795000_2025.11.02.686125v3.57","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmcl.2017.08.057"},{"key":"2026050905200795000_2025.11.02.686125v3.58","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.6b00233"},{"key":"2026050905200795000_2025.11.02.686125v3.59","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.6b00135"},{"key":"2026050905200795000_2025.11.02.686125v3.60","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2008.05.006"},{"key":"2026050905200795000_2025.11.02.686125v3.61","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.75.7.3327"},{"key":"2026050905200795000_2025.11.02.686125v3.62","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00305-09"},{"key":"2026050905200795000_2025.11.02.686125v3.63","doi-asserted-by":"publisher","DOI":"10.1128\/iai.55.3.680-685.1987"},{"key":"2026050905200795000_2025.11.02.686125v3.64","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.111.7.897"},{"key":"2026050905200795000_2025.11.02.686125v3.65","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00305-09"},{"key":"2026050905200795000_2025.11.02.686125v3.66","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00932-08"},{"key":"2026050905200795000_2025.11.02.686125v3.67","doi-asserted-by":"publisher","DOI":"10.1002\/1873-3468.70023"},{"key":"2026050905200795000_2025.11.02.686125v3.68","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-147-4-1017"},{"key":"2026050905200795000_2025.11.02.686125v3.69","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.8b00256"},{"key":"2026050905200795000_2025.11.02.686125v3.70","doi-asserted-by":"publisher","DOI":"10.1038\/nphoton.2007.251"},{"key":"2026050905200795000_2025.11.02.686125v3.71","doi-asserted-by":"publisher","DOI":"10.1038\/nm.1795"},{"key":"2026050905200795000_2025.11.02.686125v3.72","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01474-06"},{"key":"2026050905200795000_2025.11.02.686125v3.73","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-11-35"},{"key":"2026050905200795000_2025.11.02.686125v3.74","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2003.03662.x"},{"key":"2026050905200795000_2025.11.02.686125v3.75","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2001.02394.x"},{"key":"2026050905200795000_2025.11.02.686125v3.76","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.1999.01472.x"},{"key":"2026050905200795000_2025.11.02.686125v3.77","doi-asserted-by":"publisher","DOI":"10.1128\/jb.178.13.3715-3721.1996"},{"key":"2026050905200795000_2025.11.02.686125v3.78","doi-asserted-by":"publisher","DOI":"10.1128\/JB.181.3.791-798.1999"},{"key":"2026050905200795000_2025.11.02.686125v3.79","doi-asserted-by":"publisher","DOI":"10.1042\/bj3480223"},{"key":"2026050905200795000_2025.11.02.686125v3.80","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.0c07700"},{"key":"2026050905200795000_2025.11.02.686125v3.81","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0065806"},{"key":"2026050905200795000_2025.11.02.686125v3.82","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.30.18595"},{"key":"2026050905200795000_2025.11.02.686125v3.83","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.77704"},{"key":"2026050905200795000_2025.11.02.686125v3.84","doi-asserted-by":"publisher","DOI":"10.1016\/j.jlr.2022.100262"},{"key":"2026050905200795000_2025.11.02.686125v3.85","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.81924"},{"key":"2026050905200795000_2025.11.02.686125v3.86","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkg050"},{"key":"2026050905200795000_2025.11.02.686125v3.87","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-018-0048-6"},{"key":"2026050905200795000_2025.11.02.686125v3.88","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkl460"},{"key":"2026050905200795000_2025.11.02.686125v3.89","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.4c00091"},{"key":"2026050905200795000_2025.11.02.686125v3.90","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1315-z"},{"key":"2026050905200795000_2025.11.02.686125v3.91","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.48.8.3175-3178.2004"},{"key":"2026050905200795000_2025.11.02.686125v3.92","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.49.11.4775-4777.2005"},{"key":"2026050905200795000_2025.11.02.686125v3.93","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.45.3.800-804.2001"},{"key":"2026050905200795000_2025.11.02.686125v3.94","doi-asserted-by":"publisher","DOI":"10.1021\/nn4057269"},{"key":"2026050905200795000_2025.11.02.686125v3.95","doi-asserted-by":"publisher","DOI":"10.1155\/2011\/414729"},{"key":"2026050905200795000_2025.11.02.686125v3.96","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.8b06144"},{"key":"2026050905200795000_2025.11.02.686125v3.97","doi-asserted-by":"publisher","DOI":"10.1021\/ar4000554"},{"key":"2026050905200795000_2025.11.02.686125v3.98","doi-asserted-by":"publisher","DOI":"10.1110\/ps.051393805"},{"key":"2026050905200795000_2025.11.02.686125v3.99","doi-asserted-by":"publisher","DOI":"10.1021\/bi035933x"},{"key":"2026050905200795000_2025.11.02.686125v3.100","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2012.01.016"},{"key":"2026050905200795000_2025.11.02.686125v3.101","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.8b08711"},{"key":"2026050905200795000_2025.11.02.686125v3.102","doi-asserted-by":"publisher","DOI":"10.3389\/fchem.2022.921091"},{"key":"2026050905200795000_2025.11.02.686125v3.103","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200705993"},{"key":"2026050905200795000_2025.11.02.686125v3.104","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jpcb.6b05604"},{"key":"2026050905200795000_2025.11.02.686125v3.105","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.3c00565"},{"key":"2026050905200795000_2025.11.02.686125v3.106","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.02728-13"},{"key":"2026050905200795000_2025.11.02.686125v3.107","doi-asserted-by":"publisher","DOI":"10.1101\/2025.06.03.656700"},{"key":"2026050905200795000_2025.11.02.686125v3.108","doi-asserted-by":"publisher","DOI":"10.1034\/j.1399-3011.2000.00723.x"},{"key":"2026050905200795000_2025.11.02.686125v3.109","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2011.06.006"},{"key":"2026050905200795000_2025.11.02.686125v3.110","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2012.07.022"},{"key":"2026050905200795000_2025.11.02.686125v3.111","doi-asserted-by":"publisher","DOI":"10.1038\/s41557-021-00708-z"},{"key":"2026050905200795000_2025.11.02.686125v3.112","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M116.759837"},{"key":"2026050905200795000_2025.11.02.686125v3.113","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkw107"},{"key":"2026050905200795000_2025.11.02.686125v3.114","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.563030"},{"key":"2026050905200795000_2025.11.02.686125v3.115","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.9b00427"},{"key":"2026050905200795000_2025.11.02.686125v3.116","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.02183-07"},{"key":"2026050905200795000_2025.11.02.686125v3.117","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaa4690"},{"key":"2026050905200795000_2025.11.02.686125v3.118","doi-asserted-by":"publisher","DOI":"10.64898\/2026.03.19.712639"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.11.02.686125","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T12:20:17Z","timestamp":1778329217000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.11.02.686125"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,2]]},"references-count":118,"URL":"https:\/\/doi.org\/10.1101\/2025.11.02.686125","relation":{},"subject":[],"published":{"date-parts":[[2025,11,2]]},"subtype":"preprint"}}