Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability

The ability of Mycobacterium tuberculosis to persist in its human host despite extensive chemotherapy is thought to be based on subpopulations of non-replicating phenotypically drug-resistant bacilli. To study the non-growing pathogen, culture models that generate quiescent organisms by either oxygen depletion in nutrient-rich medium (Wayne model) or nutrient deprivation in oxygen-rich medium (Loebel model) have been developed. In contrast to the energy metabolism of Wayne bacilli, little is known about Loebel bacilli. Here we analysed M. tuberculosis under nutrient-starvation conditions. Upon shifting to the non-replicating state the pathogen maintained a fivefold reduced but constant intracellular ATIP level. Chemical probing of the F(0)F(1) ATIP synthase demonstrated the importance of this enzyme for ATP homeostasis and viability of the nutrient-starved organism. Surprisingly, the specific ATIP synthase inhibitor TMC207 did not affect viability and only moderately reduced the intracellular ATIP level of nutrient-starved organisms. Depletion of oxygen killed Loebel bacilli, whereas death was prevented by nitrate, suggesting that respiration and an exogenous electron acceptor are required for maintaining viability. Nutrient-starved bacilli lacking the glyoxylate shunt enzyme isocitrate lyase failed to reduce their intracellular ATIP level and died, thus establishing a link between ATIP control and intermediary metabolism. We conclude that reduction of the ATIP level might be an important step in the adaptation of M. tuberculosis to non-growing survival.