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

Abstract

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 drugresistant 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 ATP level. Chemical probing of the F 0 F 1 ATP synthase demonstrated the importance of this enzyme for ATP homeostasis and viability of the nutrient-starved organism. Surprisingly, the specific ATP synthase inhibitor TMC207 did not affect viability and only moderately reduced the intracellular ATP 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 ATP level and died, thus establishing a link between ATP control and intermediary metabolism. We conclude that reduction of the ATP level might be an important step in the adaptation of M. tuberculosis to non-growing survival.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

“…We incubated Mtb in PBS to simulate nutrient-limited conditions. Starvation in PBS confers on Mtb a high level of phenotypic tolerance to most TB drugs, including RIF, isoniazid (INH), streptomycin (STREP), moxifloxacin (MOXI), ethambutol (ETH), and bedaquiline (TMC207) (20)(21)(22). The reported 90% Loebel-cidal concentration of Mtb nutrientstarved for 14 d was 25 μM for RIF, informing the use of high doses of rifamycins for our experiments (20).…”

“…This was in question because DD Mtb generation proceeded most quickly when RIF was used at a concentration (100 μM) far above its minimal bactericidal concentration for replicating bacteria (0.01-0.08 μM) (20,21). The need for high concentrations of RIF may reflect impaired uptake of RIF by starved Mtb (21) or could be related to the phenotypic RIF resistance attributed to stress-induced protein mistranslation (39).…”

The uses and abuses of rapid bioluminescence-based ATP assays

“…In such states metabolic activity must be maintained at a very low rate. Gengenbacher et al (2010) reported that in response to nutrient starvation Mycobacterium tuberculosis was able to downregulate its ATP levels to one fifth that of normal. As a consequence of this the bacterium entered a non-replicating state, and this enabled it to resist the effects of anti-tuberculosis drugs.…”

Proteomics analysis of the response of the marine bacterium Marinobacter adhaerens HP15 to the diatom Thalassiosira weissflogii

“…While glyoxylate continues fueling the glyoxylate cycle, succinate replenishes the TCA cycle (Vanni et al 1990). The relevance of isocitrate lyase, and consequently the importance of the glyoxylate cycle in surviving nutrient-depleted conditions, had been demonstrated previously (Idnurm & Howlett 2002, Gengenbacher et al 2010. The down-regulation of a central enzyme of the glyoxylate cycle thus indicates a favorable substrate supply to the TCA cycle.…”