Triacylglycerol Utilization Is Required for Regrowth of In Vitro Hypoxic Nonreplicating Mycobacterium bovis Bacillus Calmette-Guerin

Abstract

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 ...

Lipidomic analyses of Mycobacterium tuberculosis based on accurate mass measurements and the novel “Mtb LipidDB”

“…The sum compositions indicated that the smaller TGs were probably esterifi ed with C16, C18, and C19 FAs, whereas longer C22, C24, and C26 acyl functions probably esterifi ed the larger, stationary phase-specifi c TGs. The latter observation fi ts well with previous structural reports of mycobacterial TG ( 13,32,33 ). To further support this structural information, the most-abundant logarithmic and stationary phase TG group [M+NH 4 ] + ions were targeted for collisioninduced dissociation MS/MS ( Fig.…”

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

“…We have previously shown that THL inhibits growth of M. bovis BCG and re-growth of bacilli from hypoxic NRP (11). This is also the case for the THL analog used here (supplemental Fig.…”

“…See also accumulation of large amounts of TAG was quantitatively a most prominent change in lipid composition on entry into nonreplicating conditions, and these TAG deposits were hydrolyzed during regrowth (supplemental Fig. S3A, S3B) (11). In addition, total cellular esterase activity, assessed by esterlinked fluorescent dye (FITC conjugated CMFDA, supplemental Fig.…”

“…During times of oxygen deprivation and in the absence of host cells, cultivated mycobacteria store fatty acids (FAs) in the form of triacylglycerol (TAG) 1 -enriched lipid droplets (8 -10). Upon resuscitation (by the re-introduction of oxygen), these lipid droplets vanish and TAGs are hydrolyzed (11). Unfortunately, the molecular mechanisms for TAG build-up and breakdown are far less well understood in bacteria when compared with those processes in eukaryotes.…”

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

“…Numerous components of the DosR regulon of M. smegmatis are also DosR dependent in pathogenic mycobacteria, including multiple universal stress proteins (30,31), a ribosome stabilizingfactor (32,33), and putative energy metabolism genes (e.g., diacylglycerol acyltransferases, nitroreductases) (34,35) (Tables S1 and S2). The DosR regulon of M. smegmatis also controls the expression of some genes that are not DosR dependent or are absent from the pathogen (Table S2), most notably 10 genes involved in hydrogen metabolism and 6 genes linked to sulfur metabolism ( Fig.…”