Mycobacterium tuberculosis requires the outer membrane lipid phthiocerol dimycocerosate for starvation-induced antibiotic tolerance

Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis treatment and to emergence of drug-resistant strains. Nutrient restriction induces M. tuberculosis drug tolerance, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (~1,000 mutants) by Tn-seq and identified 33 and 102 Tn mutants with altered tolerance to antibiotics in stationary phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ppgK::Tn and clpS::Tn, showed increased susceptibility to two different drug combinations in both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole genome sequencing revealed single nucleotide polymorphisms in both the ppgK::Tn and clpS::Tn mutants that prevented PDIM production. Complementation of the clpS::Tn ppsDQ291* mutant with ppsD restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening tuberculosis treatment and limiting development of drug resistance.