The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis

Abstract

The persistence of Mycobacterium tuberculosis despite prolonged chemotherapy represents a major obstacle for the control of tuberculosis. The mechanisms used by Mtb to persist in a quiescent state are largely unknown. Chemical genetic and genetic approaches were used here to study the physiology of hypoxic nonreplicating mycobacteria. We found that the intracellular concentration of ATP is five to six times lower in hypoxic nonreplicating Mtb cells compared with aerobic replicating bacteria, making them exquisitely sensitive to any further depletion. We show that de novo ATP synthesis is essential for the viability of hypoxic nonreplicating mycobacteria, requiring the cytoplasmic membrane to be fully energized. In addition, the anaerobic electron transport chain was demonstrated to be necessary for the generation of the protonmotive force. Surprisingly, the alternate ndh-2, but not -1, was shown to be the electron donor to the electron transport chain and to be essential to replenish the [NAD ؉ ] pool in hypoxic nonreplicating Mtb. Finally, we describe here the high bactericidal activity of the F0F1 ATP synthase inhibitor R207910 on hypoxic nonreplicating bacteria, supporting the potential of this drug candidate for shortening the time of tuberculosis therapy.anaerobic respiration ͉ dormancy ͉ ATP synthase ͉ NADH dehydrogenase ͉ Diarylquinoline

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

“…1 shows that the nutrient-starvation-induced shiftdown to the stationary phase was accompanied by a fivefold drop in the intracellular ATP level (from 3.39±0.21610 218 to 0.59±0.10610 218 mol c.f.u. 21 ), identical to the reduction observed for hypoxic non-replicating bacilli (Rao et al, 2008). This result suggests that ATP level reduction could be a general metabolic theme for adaptation of M. tuberculosis to quiescent survival.…”

“…Loebel bacilli require the F 0 F 1 ATP synthase to maintain ATP homeostasis and viability Previous reports demonstrated the bactericidal activity of the ATP synthase inhibitors TMC207 and DCCD against exponentially growing as well as hypoxic non-replicating bacilli (Andries et al, 2005;Koul et al, 2007;Rao et al, 2008). These findings established that the ATP synthase is essential for the survival of both physiological states of M. tuberculosis (Koul et al, 2008;Rao et al, 2008).…”

“…Recent studies of the tubercle bacillus in the Wayne culture model demonstrated that the organism reduces its intracellular ATP level fivefold when it shifts down to a hypoxic, non-replicating state (Koul et al, 2008;Rao et al, 2008). To determine whether the ATP level reduction also occurs upon the nutrient-starvation-induced shiftdown to the non-growing state, we monitored the intracellular ATP of bacilli in Loebel cultures over time.…”

“…The intracellular ATP level of Wayne bacilli is reduced fivefold compared to that of exponentially growing bacteria (Koul et al, 2008;Rao et al, 2008). The F 0 F 1 ATP synthase and the respiratory enzyme NADH dehydrogenase (Ndh)-2, but not Ndh-1, were shown to be essential for the survival of oxygen-starved bacilli (Koul et al, 2008;Rao et al, 2008). Nutrient-starved bacilli have been less well characterized.…”

“…In the current study, we have analysed the metabolism of nutrient-starved M. tuberculosis in vitro. We focused on energy metabolism, respiration and the intracellular ATP level, since these were found to be critical determinants for viability of the hypoxic non-replicating pathogen (Rao et al, 2008).…”