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 F(0)F(1) ATP synthase inhibitor R207910 on hypoxic nonreplicating bacteria, supporting the potential of this drug candidate for shortening the time of tuberculosis therapy.

Fig. 1.

ATP level is low and stable in hypoxic nongrowing mycobacteria. Dynamics of ATP concentration (filled triangles) and cfu (filled squares) during Wayne dormancy in Mtb H37Rv monitored over a period of 25 days. Mtb H37Rv cells were grown in closed test tubes with head space ratio of 0.5 and incubated at 37°C on magnetic stir platform, as described (6, 7). The times when the methylene blue (oxygen consumption indicator) exhibited noticeable fading (f) and complete decolorization (d) are shown.

Fig. 2.

F₀F₁ ATP synthase inhibitors decrease ATP concentration and viability of hypoxic nongrowing Mtb H37Rv. Hypoxic nongrowing Mtb cells were incubated with varying concentration of R207910 and DCCD over a period of 5 days at 37°C under anaerobic conditions. The ATP levels (A) and viability (B) are shown. Rifampicin (Rif) at 1 μM and metronidazole (MTZ) at 200 μM were used as controls. The experiments were carried out three times in triplicate and results are given as the mean values and standard deviations.

Fig. 3.

Aerobically growing and hypoxic nongrowing Mtb H37Rv cells have a similar membrane potential. (A) FACS analysis of DiOC₂ (3) stained hypoxic nongrowing Mtb H37Rv with or without prior treatment using 5 μM valinomycin. Shown are the mean red and green fluorescence intensities. (B) The mean fluorescence intensity ratio (red/green) of the aerobically growing (filled box) and hypoxic nongrowing (empty box) Mtb H37Rv, incubated with 30 μM DiOC2 (3) for 15 min in either the presence or absence of 5 μM valinomycin are shown.

Fig. 4.

ndh-2 is essential for survival of hypoxic nongrowing Mtb H37Rv. Varying concentrations of rotenone (ndh-1 inhibitor, 80 μM) and thioridazine (ndh-2 inhibitor) were incubated with nongrowing Mtb cells over a period of 5 days at 37°C under anaerobic conditions. The ATP levels (A) and viability (B) of nonreplicating/hypoxic Mtb H37Rv are shown. Rifampicin (Rif) at 1 μM, MTZ at 200 μM and INH at 25 μM were used as controls. The experiments were carried out three times in triplicate, and results are given as the mean values and standard deviations.

Fig. 5.

ndh-2 inhibitor reduces the membrane potential (Δψ) of hypoxic nongrowing Mtb cells. Effect of various antimycobacterial agents on the Δψ of hypoxic nongrowing Mtb H37Rv. FACS analysis showing the mean fluorescence intensity ratio (red/green) of DiOC₂ (3) stained hypoxic Mtb incubated with various drugs over a period of 5 days at 37°C under anaerobic conditions. The experiments were carried out three times in triplicate and results are given as the mean values and standard deviations.

Fig. 6.

Model of the anaerobic electron transport chain in hypoxic nonreplicating Mtb H37Rv.