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Adv."],"published-print":{"date-parts":[[2026,1,30]]},"abstract":"<jats:p>\n                    Bacterial efflux pumps are major contributors to multidrug resistance, classically described as \u201cgatekeepers\u201d that reduce drug entry. Here, we uncover a post\u2013entry mechanism of efflux pumps, revealing their function deep into intracellular drug-target interactions. Using quantitative live-cell imaging, we monitored the activity of major efflux systems in\n                    <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                    and\n                    <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                    with Hoechst 33342 (HCT), a DNA binding inhibitor. We found that inactivation of efflux (\u0394\n                    <jats:italic toggle=\"yes\">tolC<\/jats:italic>\n                    in\n                    <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                    and \u03946 in\n                    <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                    ) increased the apparent HCT-DNA affinity, mediated by a decreased apparent unbinding rate, whereas the intrinsic rate remained unchanged. Statistical physics modeling and experimental testing show that, unlike under dilute in vitro conditions, drug molecules that unbind from their targets in intracellular environments undergo successive rebinding, prolonging the total lifetime of the drug bound to the target. However, efflux pumps counteract this effect by suppressing rebinding, thereby kinetically destabilizing drug-target interactions. This biophysical mechanism acts multiplicatively with the canonical gatekeeping effect to broaden and amplify drug resistance.\n                  <\/jats:p>","DOI":"10.1126\/sciadv.aea7983","type":"journal-article","created":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T18:59:52Z","timestamp":1769626792000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":0,"title":["Efflux pumps control intracellular drug-target kinetics by limiting rebinding in bacteria"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3629-8032","authenticated-orcid":true,"given":"Subrata","family":"Dev","sequence":"first","affiliation":[{"name":"Department of Physics, Emory University, Atlanta, GA 30322, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0622-063X","authenticated-orcid":true,"given":"Keiran","family":"Stevenson","sequence":"additional","affiliation":[{"name":"Department of Physics, Emory University, Atlanta, GA 30322, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6395-6163","authenticated-orcid":true,"given":"Dai","family":"Le","sequence":"additional","affiliation":[{"name":"Department of Physics, Emory University, Atlanta, GA 30322, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1594-4971","authenticated-orcid":true,"given":"Minsu","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Physics, Emory University, Atlanta, GA 30322, USA."},{"name":"Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA."},{"name":"Antibiotic Research Center, Emory University, Atlanta, GA 30322, USA."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"221","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(21)02724-0"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro3380"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.78.082907.145923"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1080\/07853890701195262"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aar7981"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.64.4.672-693.2000"},{"key":"e_1_3_2_8_2","first-page":"215","article-title":"AcrAB and related multidrug efflux pumps of Escherichia coli","volume":"3","author":"Nikaido H.","year":"2001","unstructured":"H. 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