PROJECT SUMMARY/ABSTRACT Tuberculosis (TB), caused by the Mycobacterium tuberculosis (Mtb) complex, is the leading cause of death due to infectious disease worldwide. Treatment for drug-susceptible TB requires a multi-drug regimen prescribed for a period of six months, longer than for almost any other drug-susceptible infection. The treatment of increasingly- prevalent drug-resistant TB often demands riskier drugs taken for even longer periods of time. The length of treatment poses major challenges in terms of cost, side effects, and patient adherence. A phenomenon known as phenotypic tolerance to antibiotics, in which phenotypic differences among individual cells in a population allow some of them–known as persister cells–to survive exposure to an antibiotic against which their genomes do not encode resistance, is a main contributing factor to the protracted therapy for TB. In addition to lengthening treatment duration, persister cells also represent a source of treatment failure, contribute to TB reactivation after apparently effective treatment, and constitute a pool of surviving cells from which antibiotic-resistant strains can eventually emerge. Thus, elimination of persisters could reduce treatment times as well as cut rates of treatment failure and disease reactivation and slow emergence of drug resistance. However, in order to effectively target persisters therapeutically, a deeper understanding of how cells enter, maintain, and exit from a persistent state is required. The goal of this proposal is to dissect the molecular mechanisms underlying persistence in Mtb via two complementary approaches. In the first, the project will seek to isolate so-called high persister (hip) mutants, populations of which generate a higher proportion of persister cells than wild type strains, and characterize the molecular mechanisms by which the hip mutations they carry increase the proportion of persisters. These mutants will be isolated using a novel filter-based method that takes into account three parameters–type of antibiotic stress, antibiotic concentration, and time of exposure–to allow for the uncovering of diverse persistence mechanisms. In the second approach, the project will aim to elucidate the role of inorganic polyphosphate (poly(P)) in mycobacterial persistence. Specifically, the project will test the hypothesis that poly(P) acts as a molecular switch to control entry into and exit from a persistent state using a set of genetic knockouts in key poly(P) regulatory genes. Ultimately, this project will contribute to a molecular understanding of persistence in Mtb, which will advance efforts to target persister cells therapeutically.