Genetic and metabolic regulation of Mycobacterium tuberculosis acid growth arrest

Abstract Mycobacterium tuberculosis (Mtb) senses and adapts to acidic environments during the course of infection. Acidic pH-dependent adaptations include the induction of metabolic genes associated with anaplerosis and growth arrest on specific carbon sources. In this study, reverse and forward genetic studies were undertaken to define new mechanisms underlying pH-dependent adaptations. Here we report that deletion of isocitrate lyase ( icl1/2 ) or phosphoenolpyruvate carboxykinase ( pckA ) results in reduced growth at acidic pH and altered metabolite profiles, supporting that remodeling of anaplerotic metabolism is required for pH-dependent adaptation. Mtb cultured at pH 5.7 in minimal medium containing glycerol as a single carbon source exhibits an acid growth arrest phenotype, where the bacterium is non-replicating but viable and metabolically active. The bacterium uptakes and metabolizes glycerol and maintains ATP pools during acid growth arrest and becomes tolerant to detergent stress and the antibiotics isoniazid and rifampin. A forward genetic screen identified mutants that do not arrest their growth at acidic pH, including four enhanced acid growth ( eag ) mutants with three distinct mutations in the PPE gene MT3221. Overexpression of the MT3221(S211R) variant protein in wild type Mtb results in enhanced acid growth and reduced drug tolerance. Together, these findings provide new evidence for a genetic and physiological basis for acid growth arrest and support that growth arrest is an adaptive process and not simply a physiological limitation associated with acidic pH. Author Summary The bacterium Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis in humans. During infection Mtb colonizes a variety of environments that have acidic environments and Mtb must adapt to these environments to cause disease. One of these adaptations is that Mtb slows and arrests its growth at acidic pH, and the goal of this study was to examine the genetics and physiology of these pH-dependent adaptations. We found that Mtb modifies its metabolism at acidic pH and that these adaptations are required for optimal growth. We also found that acidic pH and specific nutrient sources can promote the bacterium to enter a state of dormancy, called acid growth arrest, where the bacterium becomes tolerant to antibiotics. Mutants were identified that do not arrest their growth at acidic, revealing that acid growth arrest is a genetically controlled process. Overall, understanding how Mtb adapts to acidic pH has revealed pathway that are required for virulence and drug tolerance and thus may identify new targets for drug development that may function to shorten the course of TB therapy.