Triacylglycerol metabolism in replicating and nonreplicating Mycobacterium bovis bacillus Calmette-Guerin

Abstract

The etiologic agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb) infects more than one-third of the world population, resulting in two million deaths annually. The success of this pathogen relies on its ability to go into a nonreplicating persisting (NRP) state upon infecting an immunocompetent human host. Intriguingly, the metabolic processes that are critical for Mtb to enter dormancy, survive in this NRP state, and reactivate upon favorable conditions are poorly characterized. Of note, hypoxia is postulated to be a key factor that triggers the NRP state in the bacilli.

Mycobacteria store large amounts of triacylglycerols (TGs), probably as energy reserves during periods of oxygen-limited stress. The kinetics and importance of TG metabolism at various different physiological states (logarithmic growth, aerated stationary phase, hypoxia-induced dormancy, regrowth from dormancy) are, however, not clearly understood.

In this study, we specifically determined the levels of TG accumulation and TG lipase activity during different metabolic states in Mycobacterium bovis bacillus Calmette-Guerin (BCG), an attenuated tubercle bacilli, closely related to Mtb. We found extensive buildup and breakdown of TGs in the bacillus during entry into and exit from hypoxic dormancy, respectively. These processes correlate with the dynamic presence of intracellular lipid inclusions. Reduction of TGs also coincides with an increase of 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 the utilization of TGs is essential for the regrowth of bacilli during exit from the hypoxic dormancy. However, enzymes involved in the anabolism and catabolism of mycobacterial TG inclusions remain relatively unknown.

Intracellular TG inclusions known as lipid droplets (LDs) are associated with the bacilli in TB patient sputum samples and hypervirulent strains. A LD comprises a hydrophobic core of mainly TGs, enveloped by phospholipid monolayer with few embedded proteins related to the metabolism of lipid inclusions. 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 identified five proteins, Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c and BCG1721, which are exclusively associated with LDs purified from hypoxic dormant M. bovis BCG. Disruption of genes tgs1, tgs2, BCG1169c, BCG1489c in mycobacteria revealed that they are indeed involved in the TG metabolic pathway. We 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, it is found that these mycobacterial proteins co-localized with host 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 in mycobacterial growth during reactivation makes this novel LD-associated factor a potential new target for therapeutic intervention.