Immunological tests provide evidence of latent tuberculosis in one third of the global population, more than two billion individuals. Latent tuberculosis is defined by the absence of clinical symptoms but carries a risk of subsequent progression to clinical disease, particularly in the context of co-infection with HIV. Here we discuss the biology of latent tuberculosis as part of a broad spectrum of responses that occur following infection with Mycobacterium tuberculosis, resulting in formation of a range of physiologically distinct granulomatous lesions that provide environments with differential ability to support or suppress persistence of viable bacteria. We go on to show how this model can be used to inform a rational programme to discover drugs that will be effective in the eradication of M. tuberculosis infection.
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
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.
PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F 420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F 420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F 420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles.comparative genome sequencing ͉ F420 ͉ nitroimidazole
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