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 (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. Human beings are the sole ecological niche for M. tuberculosis (Mtb), and it is estimated that 1.8 billion people are currently infected with Mtb. An important aspect of this infection is Mtb’s ability to maintain infection by replicating within macrophages. Within macrophages, Mtb exploits a specialized set of metabolic pathways to utilize host-derived nutrients, such as fatty acids and/or cholesterol, for energy production. Many details regarding Mtb metabolism during infection remain unknown. Here we took a chemical approach to identify small molecule probes, which target Mtb metabolism during infection in macrophages. We found that many of the small molecule inhibitors that we identified require cholesterol for activity. Here we report a novel chemical rescue approach to identify the metabolic targets of three novel inhibitors, and discovered that cAMP signaling is linked to cholesterol utilization in Mtb. Together, these data demonstrate that cholesterol exerts a dominant effect on Mtb metabolism within macrophages. Additionally, the novel inhibitors identified in this study will facilitate evaluation of cholesterol metabolism as a target for chemotherapeutic intervention.

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs.

Tuberculosis is a distinctive disease in which the causative agent, Mycobacterium tuberculosis, can persist in humans for decades by avoiding clearance from host immunity. During infection, M. tuberculosis maintains viability by extracting and utilizing essential nutrients from the host, and this is a prerequisite for all of the pathogenic activities that are deployed by the bacterium. In particular, M. tuberculosis preferentially acquires and metabolizes host-derived lipids (fatty acids and cholesterol), and the bacterium utilizes these substrates to cause and maintain disease. In this review, we discuss our current understanding of lipid utilization by M. tuberculosis, and we describe how these pathways promote pathogenesis to fuel metabolic processes in the bacillus. Finally, we highlight weaknesses in these pathways that potentially can be targeted for drug discovery.

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.