Comparing the Metabolic Capabilities of Bacteria in the Mycobacterium tuberculosis Complex

Pathogenic mycobacteria are known for their ability to maintain persistent infections in various mammals. The canonical pathogen in this genus is Mycobacterium tuberculosis and this bacterium is particularly successful at surviving and replicating within macrophages. Here, we will highlight the metabolic processes that M. tuberculosis employs during infection in macrophages and compare these findings with what is understood for other pathogens in the M. tuberculosis complex

Chewing the fat: lipid metabolism and homeostasis during M. tuberculosis infection

The interplay between Mycobacterium tuberculosis lipid metabolism, the immune response and lipid homeostasis in the host creates a complex and dynamic pathogen-host interaction. Advances in imaging and metabolic analysis techniques indicate that M. tuberculosis preferentially associates with foamy cells and employs multiple physiological systems to utilize exogenously derived fatty-acids and cholesterol. Moreover, novel insights into specific host pathways that control lipid accumulation during infection, such as the PPARgamma and LXR transcriptional regulators, have begun to reveal mechanisms by which host immunity alters the bacterial micro-environment. As bacterial lipid metabolism and host lipid regulatory pathways are both important, yet inherently complex, components of active tuberculosis, delineating the heterogeneity in lipid trafficking within disease states remains a major challenge for therapeutic design

Novel protein acetyltransferase, Rv2170, modulates carbon and energy metabolism in Mycobacterium tuberculosis

Recent data indicate that the metabolism of Mycobacterium tuberculosis (Mtb) inside its host cell is heavily dependent on cholesterol and fatty acids. Mtb exhibits a unique capacity to co-metabolize different carbon sources and the products from these substrates are compartmentalized metabolically. Isocitrate lies at one of the key nodes of carbon metabolism and can feed into either the glyoxylate shunt (via isocitrate lyase) or the TCA cycle (via isocitrate dehydrogenase (ICDH) activity) and we sought to better understand the regulation at this junction. An isocitrate lyase-deficient mutant of Mtb (Δicl1) exhibited a delayed growth phenotype in stearic acid (C18 fatty acid) media and we isolated rescue mutants that had lost this growth delay. We found that mutations in the gene rv2170 promoted Mtb replication under these conditions and rescued the growth delay in a Δicl1 background. The Mtb Rv2170 protein shows lysine acetyltransferase activity, which is capable of post-translationally modifying lysine residues of the ICDH protein leading to a reduction in its enzymatic activity. Our data show that contrary to most bacteria that regulate ICDH activity through phosphorylation, Mtb is capable of regulating ICDH activity by acetylation. This mechanism of regulation is similar to that utilized for mammalian mitochondrial ICDH

Dynamic quantitative assays of phagosomal function

Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function, to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real-time readouts of phagosomal function that enables these activities to be rigorously quantified. This chapter contains the description of several of these assays assessed by different methods of quantitation; including a Fluorescence Resonance Emission Transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometer, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measure by confocal microscope. These assays illustrate both the range parameters that can be quantified as well as the flexibility of instrumentation that can be exploited for their quantitation

Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously pro-vided evidence for the presence of two distinct LD pools, a pre-existing and a dynamic LD pool. Here we investigate the mech-anisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neu-tral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro. The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, tar-geted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa ؊/؊ mice. Lalistat partially inhibited the induction of activation marker ␣-smooth muscle actin (␣-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation

Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation

M. tuberculosis Rv2252 encodes a diacylglycerol kinase involved in the biosynthesis of phosphatidylinositol mannosides (PIMs)

Phosphorylated lipids play important roles in biological systems, not only as structural moieties but also as modulators of cellular function. Phospholipids of pathogenic bacteria are known to play roles both as membrane components and as factors that modulate the infectious process. Mycobacterium tuberculosis is, however, noteworthy in that it has an extremely diverse repertoire of biologically active phosphorylated lipids that, in the absence of a specialized protein translocation system, appear to constitute the main means of communication with the host. Many of these lipids are derived from phosphatidylinositol (PI) that is differentially processed to give rise to phosphatidylinositol mannosides (PIMs) or lipoarabinomannan. In preliminary studies on the lipid processing enzymes associated with the bacterial cell wall, a kinase activity was noted that gave rise to a novel lipid species released by the bacterium. It was determined that this kinase activity was encoded by the ORF Rv2252. Rv2252 demonstrates the capacity to phosphorylate various amphipathic lipids of host and bacterial origin, in particular a M. tuberculosis derived diacylglycerol. Targeted deletion of the rv2252 gene resulted in disruption of the production of certain higher order PIM species, suggesting a role for Rv2252 in the biosynthetic pathway of PI, a PIM precursor

V-13–009920 inhibits the 2-methylcitrate synthase PrpC.

<p><b>(A)</b> Inhibition of PrpC enzyme activity was monitored by quantifying thiol release from propionyl-CoA leading to the formation of 3-thio-6-nitrobenzoate from DTNB measured at 412 nm. V-13–009920 inhibits pure PrpC enzyme with an IC₅₀ value of 4.0 ± 1.1 uM. (<b>B</b>) Chemical structure of V-13–009920 (5-(4-chlorophenyl)-<i>N</i>-(4-(<i>N</i>-(5-methyl-1,3,4-thiadiazol-2-yl)sulfamoyl)phenyl)-2-(trifluoromethyl)furan-3-carboxamide. Data are representative of two independent experiments.</p

Novel Inhibitors of Cholesterol Degradation in <i>Mycobacterium tuberculosis</i> Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

<div><p><i>Mycobacterium tuberculosis</i> (Mtb) relies on a specialized set of metabolic pathways to support growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medium containing cholesterol as the principle carbon source. Based on this observation, we developed a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also revealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol.</p></div