Brian C. VanderVen

Intracellular Mycobacterium tuberculosis Exploits Host-derived Fatty Acids to Limit Metabolic Stress

Background: The metabolism of host lipids is central to the survival of intracellular Mtb. Results: Degradation of cholesterol generates propionyl-CoA, which is, in part, detoxified through incorporation into methyl-branched lipids. Conclusion: The balance of acetyl-CoA versus propionyl-CoA concentrations in Mtb impacts detoxification of propionyl-CoA. Significance: The ability of Mtb to utilize host-derived lipids effectively is key to its success as a pathogen. Recent data indicate that the nutrients available to Mycobacterium tuberculosis (Mtb) inside its host cell are restricted in their diversity. Fatty acids and cholesterol appear more favored; however, their degradation can result in certain metabolic stresses. Their breakdown can generate propionyl-CoA, which gives rise to potentially toxic intermediates. Detoxification of propionyl-CoA relies on the activity of the methylcitrate cycle, the methylmalonyl pathway, or incorporation of the propionyl-CoA into methyl-branched lipids in the cell wall. The current work explores carbon flux through these pathways, focusing primarily on those pathways responsible for the incorporation of propionyl-CoA into virulence-associated cell wall lipids. Exploiting both genetic and biochemical rescue, we demonstrate that these metabolic pressures are experienced by Mtb inside its host macrophage and that the bacterium accesses host fatty acid stores. The metabolism of these host lipids expands the acetyl-CoA pool and alleviates the pressure from propionyl-CoA. These data have major implications for our appreciation of central metabolism of Mtb during the course of infection.

Mycobacterium tuberculosis Wears What It Eats

Mycobacterium tuberculosis remains one of the most pernicious of human pathogens. Current vaccines are ineffective, and drugs, although efficacious, require prolonged treatment with constant medical oversight. Overcoming these problems requires a greater appreciation of M. tuberculosis in the context of its host. Upon infection of either macrophages in culture or animal models, the bacterium realigns its metabolism in response to the new environments it encounters. Understanding these environments, and the stresses that they place on M. tuberculosis, should provide insights invaluable for the development of new chemo- and immunotherapeutic strategies.

The macrophage marches on its phagosome: dynamic assays of phagosome function

Professional phagocytes ingest particulate material to fulfil a diverse array of functions in a multicellular organism. The ancestral function of phagosomes is digestion; however, through evolution this degradative capacity has become pivotal to the adaptive immune response by processing antigens to be presented to lymphocytes. Moreover, phagocytes have also acquired an active role in microbial killing. This Innovation article describes new assays that probe the biological activities which occur within phagosomes. These assays provide functional insights into how the phagosome fulfils its diverse roles in homeostasis and in innate and adaptive immune responses.

Novel inhibitors of cholesterol degradation in Mycobacterium tuberculosis reveal how the bacterium's metabolism is constrained by the intracellular environment.

Mycobacterium tuberculosis (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.

Intraphagosomal Measurement of the Magnitude and Duration of the Oxidative Burst

Generation of an oxidative burst within the phagosomes of neutrophils, dendritic cells and macrophages is an essential component of the innate immune system. To examine the kinetics of the oxidative burst in the macrophage phagosome, we developed two new assays using beads coated with oxidation‐sensitive fluorochromes. These assays permitted quantification and temporal resolution of the oxidative burst within the phagosome. The macrophage phagosomal oxidative burst is short lived, with oxidation of bead‐associated substrates reaching maximal activity within 30 min following phagocytosis. Additionally, the extent and rate of macrophage phagosomal substrate oxidation were subject to immunomodulation by activation with lipopolysaccharide and/or interferon‐γ.

Pathway Profiling in Mycobacterium tuberculosis ELUCIDATION OF CHOLESTEROL-DERIVED CATABOLITE AND ENZYMES THAT CATALYZE ITS METABOLISM

Background: Cholesterol metabolism is critical in the chronic phase of Mycobacterium tuberculosis infection. Results: A cholesterol metabolite structure and an enzyme activity responsible for its degradation were determined. Conclusion: The igr operon encodes the enzymes that catalyze the final three steps in cholesterol side-chain degradation. Significance: Insight into the function of enzymes encoded in the igr operon is important for understanding the role of cholesterol metabolism in pathogenesis. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-14C]cholesterol or [1,7,15,22,26-13C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2′-propanoate)-3aα-H-4α-(3′-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2′-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2′-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites.

Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

Russell et al. show that activation of Mycobacterium tuberculosis–infected macrophages in vitro and in vivo enhances drug tolerance and renders the bacilli more refractory to drug-dependent killing.

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 PPARγ 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.

Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis

Pathogenic bacteria have evolved highly specialized systems to extract essential nutrients from their hosts. Mycobacterium tuberculosis (Mtb) scavenges lipids (cholesterol and fatty acids) to maintain infections in mammals but mechanisms and proteins responsible for the import of fatty acids in Mtb were previously unknown. Here, we identify and determine that the previously uncharacterized protein Rv3723/LucA, functions to integrate cholesterol and fatty acid uptake in Mtb. Rv3723/LucA interacts with subunits of the Mce1 and Mce4 complexes to coordinate the activities of these nutrient transporters by maintaining their stability. We also demonstrate that Mce1 functions as a fatty acid transporter in Mtb and determine that facilitating cholesterol and fatty acid import via Rv3723/LucA is required for full bacterial virulence in vivo. These data establish that fatty acid and cholesterol assimilation are inexorably linked in Mtb and reveals a key function for Rv3723/LucA in in coordinating thetransport of both these substrates. DOI: http://dx.doi.org/10.7554/eLife.26969.001

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.