Mycobacteria store triacylglycerols (TGs) under various stress conditions, such as hypoxia, exposure to nitric oxide, and acidic environments. These stress conditions are known to induce nonreplicating persistence in mycobacteria. The importance of TG accumulation and utilization during regrowth is not clearly understood. Here we specifically determined the levels of accumulated TG and TG lipase activity in Mycobacterium bovis bacillus Calmette-Guerin (BCG) in various different physiological states (logarithmic growth, aerated stationary phase, hypoxia-induced dormancy, and regrowth from dormancy). We found extensive accumulation and degradation of TGs in the bacilli during entry into and exit from hypoxia-induced dormancy, respectively. These processes are accompanied by dynamic appearance and disappearance of intracellular TG lipid particles. The reduction in TG levels coincides with an increase in cellular TG lipase activity in the regrowing bacilli. Tetrahydrolipstatin, an inhibitor of TG lipases, reduces total lipase activity, prevents breakdown of TGs, and blocks the growth of mycobacteria upon resuscitation with air. Our results demonstrate that utilization of TGs is essential for the regrowth of mycobacteria during their exit from the hypoxic nonreplicating state.Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the major infectious diseases, which affects one-third of the world's population (http://www.who .int/mediacentre/factsheets/fs104/en/). The majority of TB patients carry a latent infection. However, reactivation leading to active disease (16, 27) often occurs once the host defenses are weakened. During the latency period, mycobacteria are tolerant to many conventional antibiotics (23, 28), thus making eradication of TB challenging.In the human body, M. tuberculosis is believed to persist in lung lesions with hypoxic environments (6, 27). Wayne and Hayes established an "in vitro dormancy model" in which mycobacterial cultures are subjected to gradual depletion of oxygen, which causes the obligate aerobic cells to exit the cell cycle and enter into a nonreplicating persistent state (26). The bacilli in the nonreplicating persistent state are phenotypically drug resistant. Recent efforts to explore the mechanisms which allow the tubercle bacilli to enter into dormancy and survive in the host for a long period of time suggest that fatty acids (FAs) could be an important source of energy during the persistent state (1,17,20). In particular, triacylglycerols (TGs), a class of neutral lipids, are postulated to be a likely source of FAs (8).TGs are an efficient form of energy reserves in many organisms during long-term survival.Recently, it was reported that tubercle bacilli in sputum from patients with TB contain lipid bodies (11). Moreover, TGs accumulate in hypervirulent W-Beijing strains of M. tuberculosis (19). It is interesting that TGs accumulate in these clinical strains of TB. However, no systematic study has been carried out yet to investigate the accumulation and ...
Mycobacteria store triacylglycerols (TGs) in the form of intracellular lipid droplets (LDs) during hypoxia-induced nonreplicating persistence. These bacteria are phenotypically drug-resistant and therefore are believed to be the cause for prolonged tuberculosis treatment. LDs are also associated with bacilli in tuberculosis patient sputum and hypervirulent strains. Although proteins bound to LDs are well characterized in eukaryotes, the identities and functions of such proteins have not been described in mycobacteria. Here, we have identified five proteins: Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c, and BCG1721, which are exclusively associated with LDs purified from hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guérin (BCG). Disruption of genes tgs1, tgs2, BCG1169c, and BCG1489c in M. bovis BCG revealed that they are indeed involved in TG metabolism. We also characterized BCG1721, an essential bi-functional enzyme capable of promoting buildup and hydrolysis of TGs, depending on the metabolic state. Nonreplicating mycobacteria overexpressing a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TG breakdown and regrowth upon resuscitation. In addition, by heterologous expression in baker's yeast, these mycobacterial proteins also co-localized with LDs and complemented a lipase-deficient yeast strain, indicating that neutral lipid deposition and homeostasis in eukaryotic and prokaryotic microorganisms are functionally related. The demonstrated functional role of BCG1721 to support growth upon resuscitation makes this novel LD-associated factor a potential new target for therapeutic intervention.Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) 5 infects more than one-third of the world population, resulting in two million deaths per year. The success of this pathogen hinges on its ability to enter into a nonreplicating persisting (NRP) state upon infecting the human host. In this state, the mycobacteria survive quiescently for decades (1) and reactivate opportunistically, such as during co-infection with human immunodeficiency virus, to cause disease (2). However, metabolic processes and enzymes necessary for the tubercle bacilli to transit into and out of the dormant state are not well characterized yet.It is widely recognized that M. tuberculosis utilizes fatty acids (FAs) as a carbon source during persistent infection (3). Mycobacteria store FAs in the form of triacylglycerols (TGs) during dormancy (4, 5) and in TB patient sputum (6, 7). Moreover, a direct correlation between the buildup of TGs and phenotypic drug resistance in M. tuberculosis was established (5), highlighting the importance of these lipids during latency and pathogenicity. TG utilization occurs when M. tuberculosis is subjected to a nutrient-deprived environment (8) and when Mycobacterium bovis bacillus Calmette-Guérin (BCG) is resuscitated from a hypoxic, nongrowing state (9), which underscores the important functional role of intracellular lipid storage during NRP...
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