Despite a slight decline since 2014, tuberculosis (TB) remains the major deadly infectious disease worldwide with about 1.5 million deaths each year and with about one-third of the population being latently infected with Mycobacterium tuberculosis, the etiologic agent of TB. During primo-infection, the recruitment of immune cells leads to the formation of highly organized granulomas. Among the different cells, one outstanding subpopulation is the foamy macrophage (FM), characterized by the abundance of triacylglycerol-rich lipid bodies (LB). M. tuberculosis can reside in FM, where it acquires, from host LB, the neutral lipids which are subsequently processed and stored by the bacilli in the form of intracytosolic lipid inclusions (ILI). Although host LB can be viewed as a reservoir of nutrients for the pathogen during latency, the molecular mechanisms whereby intraphagosomal mycobacteria interact with LB and assimilate the LB-derived lipids are only beginning to be understood. Past studies have emphasized that these physiological processes are critical to the M. tuberculosis infectious-life cycle, for propagation of the infection, establishment of the dormancy state and reactivation of the disease. In recent years, several animal and cellular models have been developed with the aim of dissecting these complex processes and of determining the nature and contribution of their key players. Herein, we review some of the in vitro and in vivo models which allowed to gain significant insight into lipid accumulation and consumption in M. tuberculosis, two important events that are directly linked to pathogenicity, granuloma formation/maintenance and survival of the tubercle bacillus under non-replicative conditions. We also discuss the advantages and limitations of each model, hoping that this will serve as a guide for future investigations dedicated to persistence and innovative therapeutic approaches against TB.

Mycobacteria share with other actinomycetes the ability to produce large quantities of triacylglycerol (TAG), which accumulate as intracytoplasmic lipid inclusions (ILI) also known as lipid droplets (LD). Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis, acquires fatty acids from the human host which are utilized to synthesize TAG, subsequently stored in the form of ILI to meet the carbon and nutrient requirements of the bacterium during long periods of persistence. However, environmental factors governing mycobacterial ILI formation and degradation remain poorly understood. Herein, we demonstrated that in the absence of host cells, carbon excess and nitrogen starvation promote TAG accumulation in the form of ILI in M. smegmatis and M. abscessus, used as surrogate species of M. tb. Based on these findings, we developed a simple and reversible in vitro model to regulate ILI biosynthesis and hydrolysis in mycobacteria. We also showed that TAG formation is tgs1 dependent and that lipolytic enzymes mediate TAG breakdown. Moreover, we confirmed that the nitrogen-deprived and ILI-rich phenotype was associated with an increased tolerance towards several drugs used for treating mycobacterial infections. Importantly, we showed that the presence of ILI substantially enhanced the bacterial burden and granuloma abundance in zebrafish embryos infected with lipid-rich M. abscessus as compared to embryos infected with lipid-poor M. abscessus, suggesting that ILI are actively contributing to mycobacterial virulence and pathogenesis.

We still do not completely understand why tuberculosis (TB) treatment requires the combination of several antibiotics for up to 6 months. M. tuberculosis is a facultative intracellular pathogen, and it is still unknown whether heterogenous and dynamic intracellular populations of bacteria in different cellular environments affect antibiotic efficacy. By developing a dual live imaging approach to monitor mycobacterial pH homeostasis, host cell environment, and antibiotic action, we show here that intracellular localization of M. tuberculosis affects the efficacy of one first-line anti-TB drug.

Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses. However, the cellular mechanisms by which peroxisomes contribute to bacterial elimination in macrophages remain elusive. Here, we investigated peroxisome function in iPSC-derived human macrophages (iPSDM) during infection with Mycobacterium tuberculosis (Mtb). We discovered that Mtb-triggered peroxisome biogenesis requires the ESX-1 type 7 secretion system, critical for cytosolic access. iPSDM lacking peroxisomes were permissive to Mtb wild-type (WT) replication but were able to restrict an Mtb mutant missing functional ESX-1, suggesting a role for peroxisomes in the control of cytosolic but not phagosomal Mtb. Using genetically encoded localization-dependent ROS probes, we found peroxisomes increased ROS levels during Mtb WT infection. Thus, human macrophages respond to the infection by increasing peroxisomes that generate ROS primarily to restrict cytosolic Mtb. Our data uncover a peroxisome-controlled, ROS-mediated mechanism that contributes to the restriction of cytosolic bacteria.

Tuberculosis caused by Mycobacterium tuberculosis is currently one of the leading causes of death from an infectious agent. The main difficulties encountered in eradicating this bacteria are mainly related to (i) a very complex lipid composition of the bacillus cell wall, (ii) its ability to hide from the immune system inside the granulomas, and (iii) the increasing number of resistant strains. In this context, we were interested in the Rv0646c (lipG_MTB) gene located upstream to the mmaA cluster which is described as being crucial for the production of cell wall components and required for the bacilli adaptation and survival in mouse macrophages. Using biochemical experiments combined with the construction of deletion and overexpression mutant strains in Mycobacterium smegmatis, we found that LipG_MTB is a cytoplasmic membrane-associated enzyme that displays both phospholipase and thioesterase activities. Overproduction of LipG_MTB decreases the glycopeptidolipids (GPL) level concomitantly to an increase in phosphatidylinositol (PI) which is the precursor of the PI mannoside (PIM), an essential lipid component of the bacterial cell wall. Conversely, deletion of the lipG_MS gene in M. smegmatis leads to an overproduction of GPL, and subsequently decreases the strain susceptibility to various antibiotics. All these findings demonstrate that LipG is involved in cell envelope biosynthesis/remodeling, and consequently this enzyme may thus play an important role in mycobacterial physiology.