Functional Whole Genome Screen of Nutrient-Starved Mycobacterium tuberculosis Identifies Genes Involved in Rifampin Tolerance

Abstract

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the causative agent of tuberculosis (TB), poses a global health challenge and is responsible for over a million deaths each year. Current treatment is lengthy and complex, and new, abbreviated regimens are urgently needed. <i>Mtb</i> adapts to nutrient starvation, a condition experienced during host infection, by shifting its metabolism and becoming tolerant to the killing activity of bactericidal antibiotics. An improved understanding of the mechanisms mediating antibiotic tolerance in <i>Mtb</i> can serve as the basis for developing more effective therapies. We performed a forward genetic screen to identify candidate <i>Mtb</i> genes involved in tolerance to the two key first-line antibiotics, rifampin and isoniazid, under nutrient-rich and nutrient-starved conditions. In nutrient-rich conditions, we found 220 mutants with differential antibiotic susceptibility (218 in the rifampin screen and 2 in the isoniazid screen). Following <i>Mtb</i> adaptation to nutrient starvation, 82 mutants showed differential antibiotic susceptibility (80 in the rifampin screen and 2 in the isoniazid screen). Using targeted mutagenesis, we validated the rifampin-hypersusceptible phenotype under nutrient starvation in <i>Mtb</i> mutants lacking the following genes: <i>ercc3</i>, <i>moeA1</i>, <i>rv0049</i>, and <i>rv2179c</i>. These findings shed light on potential therapeutic targets, which could help shorten the duration and complexity of antitubercular regimens.