The bacterial pH gradient contributes to persistence in Mycobacterium tuberculosis

Tuberculosis (TB) remains difficult to cure due in part to poorly defined drug-tolerant persister cells formed by Mycobacterium tuberculosis (Mtb), which survive antibiotic treatment without evidence of genetic resistance. To better define this phenotype, we screened 2,336 U.S. Food and Drug Administration-approved drugs for compounds that target persistence. Unexpectedly, we identified a strong inducer of drug tolerance-the antiparasitic niclosamide (NCA), which is known to disrupt proton motive force. In contrast to earlier reports that it harbors promising anti-TB activity, we found that NCA protected Mtb from bactericidal doses of isoniazid, rifampicin, and other standard TB drugs. Investigating further, we showed that disruption of the pH gradient and consequent intracellular acidification is needed to induce tolerance, while disruption of membrane potential is not, and also that protection is tunable by external pH. Transcriptomic analysis of these chemically induced persister cells implicated Mtb-specific genes in this phenotype, and targeted knockdowns confirmed roles for three genes in either promoting or mitigating the tolerant state. These findings highlight that chemical disruption of the pH gradient is a facile and rapid means to induce drug tolerance, offering a potentially useful tool to probe persister biology in TB and other infectious diseases.IMPORTANCETuberculosis (TB) is difficult to cure due in part to poorly defined drug-tolerant persister cells formed by Mycobacterium tuberculosis (Mtb) that survive antibiotic treatment without genetic resistance. In this work, we screened for drugs that target persistence. Unexpectedly, we identified a strong inducer of drug tolerance-the antiparasitic agent niclosamide (NCA), which is known to disrupt proton motive force. We found that NCA protected Mtb from lethal doses of most standard TB drugs. We showed that disruption of the pH gradient and consequent intracellular acidification is needed to induce tolerance, and that protection is tunable by external pH. We identified three Mtb genes that contribute to either promoting or mitigating the tolerant state. These findings highlight that chemical disruption of the pH gradient is a facile and rapid way to induce drug tolerance, offering a potentially useful tool to probe persister biology in TB and other infectious diseases.