Pulmonary tuberculosis (TB) exhibits granulomatous inflammation, a site of controlling bacterial dissemination at the cost of host tissue damage. Intrigued by the granuloma type-dependent expression of inflammatory markers in TB, we sought to investigate underlying metabolic changes that drive amplification of inflammation in TB. Here, we show an association of higher inflammation in necrotic granulomas with the presence of triglyceride (TG)-rich foamy macrophages. The conspicuous absence of these macrophages in solid granulomas identified a link between the ensuing pathology and the metabolic programming of foamy macrophages. Consistent with in vivo findings, in vitro infection of macrophages with Mycobacterium tuberculosis (Mtb) led to increase in TG synthesis only under conditions of ~60% necrosis. Genetic and pharmacologic intervention that reduced necrosis prevented this bystander response. We further demonstrate that necrosis independent of Mtb also elicits the same bystander response in human macrophages. We identified a role for the human enzyme involved in TG synthesis, diacylglycerol O-acyltransferase (DGAT1), in this phenomenon. The increased TG levels in necrosis-associated foamy macrophages promoted the pro-inflammatory state of macrophages to infection while silencing expression of diacylglycerol O-acyltransferase (DGAT1) suppressed expression of pro-inflammatory genes. Our data thus invoke a role for storage lipids in the heightened host inflammatory response during infection-associated necrosis. Our data provide a functional role to macrophage lipid droplets in host defense and open new avenues for developing host-directed therapies against TB.

The mycobacterial cell wall is a chemically complex array of molecular entities that dictate the pathogenesis of Mycobacterium tuberculosis. Biosynthesis and maintenance of this dynamic entity in mycobacterial physiology is still poorly understood. Here we demonstrate a requirement for M. tuberculosis MmpL11 in the maintenance of the cell wall architecture and stability in response to surface stress. In the presence of a detergent like Tyloxapol, a mmpL11 deletion mutant suffered from a severe growth attenuation as a result of altered membrane polarity, permeability and severe architectural damages. This mutant failed to tolerate permissible concentrations of cis-fatty acids suggesting its increased sensitivity to surface stress, evident as smaller colonies of the mutant outgrown from lipid rich macrophage cultures. Additionally, loss of MmpL11 led to an altered cellular fatty acid flux in the mutant: reduced incorporation into membrane cardiolipin was associated with an increased flux into the cellular triglyceride pool. This increase in storage lipids like triacyl glycerol (TAG) was associated with the altered metabolic state of higher dormancy-associated gene expression and decreased sensitivity to frontline TB drugs. This study provides a detailed mechanistic insight into the function of mmpL11 in stress adaptation of mycobacteria.

Growing evidence suggests the importance of lipid metabolism in pathogenesis of tuberculosis. Neutral lipids form the majority of lipids in a caseous granuloma, a pathology characteristic of tuberculosis. Cytosolic lipid droplets (LDs) of macrophages form the store house of these lipids and have been demonstrated to contribute to the inflammatory response to infection. The proteome of lipid droplets reflects the mechanisms of lipid metabolism active under a condition. However, infection induced changes in the proteome of these dynamic organelles remains elusive. Here, we employed quantitative proteomics to identify alterations induced upon infection with live Mycobacterium tuberculosis (Mtb) in comparison with heat killed bacilli or uninfected macrophages. We found increased abundance of proteins coupled with lipid metabolism, protein synthesis, and vesicular transport function in LDs upon infection with live Mtb. Using biochemical methods and microscopy, we validated ADP-ribosyltransferase (Arf)-like 8 (ARL8B) to be increased on the lipid droplet surface of live Mtb infected macrophages and that ARL8B is a bonafide LD protein. This study provides the first proteomic evidence that the dynamic responses to infection also encompass changes at the level of LDs. This information will be important in understanding how Mtb manipulates lipid metabolism and defense mechanisms of the host macrophage.