Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability Martin Gengenbacher, Srinivasa P. S. Rao, Kevin Pethe and Thomas Dick Correspondence Martin Gengenbacher martin.gengenbacher@ novartis.com Novartis Institute for Tropical Diseases Pte Ltd, 10 Biopolis Road, # 05-01 Chromos, 138670, Singapore Received 27 July 2009 Revised 23 September 2009 Accepted 1 October 2009 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 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. INTRODUCTION Mycobacterium tuberculosis claims approximately 2 million lives annually (Harries & Dye, 2006). A major objective of ongoing drug discovery efforts is to shorten chemotherapy from the current 6 months to 2 months or less (Williams & Duncan, 2007). Popular working models suggest that unfavourable micro-environmental conditions inside the human lesions, such as oxygen and nutrient limitation, are external triggers that terminate growth of subpopulations of tubercle bacilli and render them phenotypically resistant to drugs, thus explaining the long treatment periods needed to cure the disease (Dick, 2001; Wayne & Sohaskey, 2001). Indeed, in vitro models using oxygen depletion in nutrient-rich medium (Wayne model) or nutrient star- vation in oxygen-rich medium (Loebel model) have demonstrated the survival of M. tuberculosis for extended periods in a non-replicating and drug-tolerant state (Betts et al., 2002; Wayne & Hayes, 1996). Recently, the energy metabolism of hypoxic non-replicating tubercle bacilli has been characterized. 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. Loebel et al. (1933a) showed that survival of the tubercle bacillus under those conditions was accompanied by a drastic reduction of respiration, indicating a very low metabolic rate or even metabolic inactivity. This was further supported by more recent transcriptomic analyses, which demonstrated a strong downregulation of metabolic genes, including those encoding enzymes of the respiratory machinery (Betts et al., 2002). Animal models as well as human lesion studies provided evidence for the presence of Loebel-like bacilli in vivo: the stringent response essential for adaptation to nutrient starvation in vitro (Betts et al., 2002; Primm et al., 2000) was shown to be required for persistence of bacilli in Abbreviations: DCCD, N,N9-dicyclohexylcarbodiimide; Icl, isocitrate lysase; Ndh, NADH dehydrogenase; r.l.u., relative light unit. A supplementary figure is available with the online version of this paper. Microbiology (2010), 156, 81–87 DOI 10.1099/mic.0.033084-0 033084 G 2010 Novartis Institute for Tropical Diseases Pte Ltd Printed in Great Britain 81