Genetic determinants of <i>Mycobacterium tuberculosis</i> adaptation and drug efficacy during stationary phase growth

The adaptation of <i>Mycobacterium tuberculosis</i> (Mtb) to a slowly growing or nongrowing state in growth-limited conditions plays a crucial role for drug tolerance. Although the mechanisms of Mtb adaptation under growth-limited conditions have been extensively studied, it remains unclear to what extent the cellular processes necessary to sustain nongrowing state affect drug efficacy. To investigate this, we performed a genome-wide transposon mutant screen, which allowed parallel identification of the genes that influence bacterial fitness and drug efficacy during the stationary phase. Our analysis revealed that genes encoding the SOS response, membrane phospholipid biosynthesis, proteasomal protein degradation, and cell wall remodeling critically determine Mtb fitness in both stationary-phase condition and antibiotic exposure. Surprisingly, we found that many mutants that compromise stationary-phase adaptation result in increased fitness during antibiotic treatment, including the recently identified genetic markers associated with poor clinical outcomes. Furthermore, genes involved in cell envelope biosynthesis and remodeling, antibiotic efflux, and phosphate transport are significantly enriched in the mutants sensitized to antibiotics, indicating that reduced drug entry is a critical factor that limits antibiotic efficacy in nonreplicating Mtb. We demonstrated that mutants deficient in utilization of lipids, the primary carbon sources for Mtb during infection, became tolerant to killing by rifampicin. We provided genetic and metabolic evidence that the activities of lipid metabolism are associated with rifampicin efficacy. These findings provide the detailed assessment of Mtb genes necessary for adaptation to the stationary phase and drug treatment and new insights into the mechanisms of antibiotic tolerance in nongrowing Mtb.IMPORTANCEIt has long been known that antibiotic efficacy is generally proportional to the bacterial growth rate. Yet it remains unclear how and to what extent the growth arrest-induced physiological and metabolic changes affect drug efficacy. Using the genome-wide transposon mutant screen, we identified the mutants that influence <i>Mycobacterium tuberculosis</i> adaptation and drug efficacy during the stationary phase of growth. We revealed both positive and negative correlations between stationary phase adaptation and drug sensitivity and identified many mutants that compromise stationary phase adaptation and result in increased fitness during antibiotic treatment, including the identified genetic markers associated with poor clinical outcomes. These results provide new insights into the mechanisms of antibiotic tolerance in nongrowing Mtb and suggest potential targets for drug development.