The 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 FDA-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 (CIP) cells implicated specific genes in this phenotype, and targeted knockdowns confirmed roles for three genes in either promoting or mitigating the tolerance 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.