Lipid Droplet-associated Proteins Are Involved in the Biosynthesis and Hydrolysis of Triacylglycerol in Mycobacterium bovis Bacillus Calmette-Guérin

Abstract

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...

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis

“…Furthermore, pathogenic Mycobacterium species usurp and hydrolyze TAG from host LDs within foamy macrophages, incorporating the resulting fatty acid products into its own LDs for use as an energy source during NRP (58,69,71). Consistent with this observation, Mycobacterium bovis BCG strains that are inhibited in their ability to mediate TAG hydrolysis are attenuated for survival in vitro during and following resuscitation from NRP (60,61). Similarly, an M. tuberculosis mutant deleted in triacylglycerol-1 synthase (⌬tgs1), an enzyme used to make TAGs, is compromised in its ability to accumulate intracellular TAGs and is unable to enter into NRP in an in vitro human TB granuloma model (73).…”

“…LD production is induced in M. tuberculosis under conditions that also promote the establishment of NRP (56). Furthermore, it has been shown that M. tuberculosis mobilizes TAGs contained within LDs as an energy source during NRP (58)(59)(60)(61). To determine whether Rv2744c regulated the survival of M. tuberculosis during NRP, wild-type H37Rv, the ⌬Rv2744c mutant, and the ⌬Rv2744c variant overproducing Rv2744c were assessed using the rapid anaerobic dormancy (RAD) model (39).…”

The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans

“…For example, mammalian PLIN 1, 2, and 3 can be targeted to bacterial lipid droplets ( 99 ), and Drosophila Lsd1 and Lsd2 can be targeted to the lipid droplets of CHO cells ( 100 ). Furthermore, Dictyostelium Lsd1 can be targeted to CHO cell lipid droplets ( 100 ), and fi ve mycobacterial proteins can be targeted to yeast lipid droplets ( 43 ). Therefore, it appears that these lipid droplet structural proteins from diverse species have certain properties in common that allow them to be properly targeted to lipid droplets.…”

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

“…A family of 24 carboxyl ester hydrolases called "lip" genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15)(16)(17)(18).…”