MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis.

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis.

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3', 5'-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

Cyclic AMP-Mediated Inhibition of Cholesterol Catabolism in Mycobacterium tuberculosis by the Novel Drug Candidate GSK2556286.

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis, shortens treatment in rodents relative to standard of care drugs. Moreover, GSK286 can replace linezolid in the three-drug, Nix-TB regimen. Given its therapeutic potential, we sought to better understand the mechanism of action of GSK286. The compound blocked growth of M. tuberculosis in cholesterol media and increased intracellular cAMP levels ~50-fold. GSK286 did not inhibit growth of an rv1625c transposon mutant in cholesterol media and did not induce cyclic AMP (cAMP) production in this mutant, suggesting that the compound acts on this adenylyl cyclase. GSK286 also induced cAMP production in Rhodococcus jostii RHA1, a cholesterol-catabolizing actinobacterium, when Rv1625c was heterologously expressed. However, these elevated levels of cAMP did not inhibit growth of R. jostii RHA1 in cholesterol medium. Mutations in rv1625c conferred cross-resistance to GSK286 and the known Rv1625c agonist, mCLB073. Metabolic profiling of M. tuberculosis cells revealed that elevated cAMP levels, induced using either an agonist or a genetic tool, did not significantly affect pools of steroid metabolites in cholesterol-incubated cells. Finally, the inhibitory effect of agonists was not dependent on the N-acetyltransferase MtPat. Together, these data establish that GSK286 is an Rv1625c agonist and sheds light on how cAMP signaling can be manipulated as a novel antibiotic strategy to shorten TB treatments. Nevertheless, the detailed mechanism of action of these compounds remains to be elucidated.

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.

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.

2-N-Arylthiazole inhibitors of Mycobacterium tuberculosis.

To develop agents for the treatment of infections caused by Mycobacterium tuberculosis, a novel phenotypic screen was undertaken that identified a series of 2-N-aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis. Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro. The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo.

Intracellular Mycobacterium tuberculosis exploits host-derived fatty acids to limit metabolic stress.

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.

Exploiting cAMP signaling in Mycobacterium tuberculosis for drug discovery.

Mycobacterium tuberculosis (Mtb) replicates within host macrophages by adapting to the stressful and nutritionally constrained environments in these cells. Exploiting these adaptations for drug discovery has revealed that perturbing cAMP signaling can restrict Mtb growth in macrophages. Specifically, compounds that agonize or stimulate the bacterial enzyme, Rv1625c/Cya, induce cAMP synthesis and this interferes with the ability of Mtb to metabolize cholesterol. In murine tuberculosis (TB) infection models, Rv1625c/Cya agonists contribute to reducing relapse and shortening combination treatments, highlighting the therapeutic potential for this class of compounds. More recently, cAMP signaling has been implicated in regulating fatty acid utilization by Mtb. Thus, a new model is beginning to emerge in which cAMP regulates the utilization of host lipids by Mtb during infection, and this could provide new targets for TB drug development. Here, we summarize the current understanding of cAMP signaling in Mtb with a focus on our understanding of how cAMP signaling impacts Mtb physiology during infection. We also discuss additional cAMP-related drug targets in Mtb and other bacterial pathogens that may have therapeutic potential.

Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis.

Cyclic AMP (cAMP) is a ubiquitous second messenger that transduces signals from cellular receptors to downstream effectors. Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, devotes a considerable amount of coding capacity to produce, sense, and degrade cAMP. Despite this fact, our understanding of how cAMP regulates Mtb physiology remains limited. Here, we took a genetic approach to investigate the function of the sole essential adenylate cyclase in Mtb H37Rv, Rv3645. We found that a lack of rv3645 resulted in increased sensitivity to numerous antibiotics by a mechanism independent of substantial increases in envelope permeability. We made the unexpected observation that rv3645 is conditionally essential for Mtb growth only in the presence of long-chain fatty acids, a host-relevant carbon source. A suppressor screen further identified mutations in the atypical cAMP phosphodiesterase rv1339 that suppress both fatty acid and drug sensitivity phenotypes in strains lacking rv3645. Using mass spectrometry, we found that Rv3645 is the dominant source of cAMP under standard laboratory growth conditions, that cAMP production is the essential function of Rv3645 in the presence of long-chain fatty acids, and that reduced cAMP levels result in increased long-chain fatty acid uptake and metabolism and increased antibiotic susceptibility. Our work defines rv3645 and cAMP as central mediators of intrinsic multidrug resistance and fatty acid metabolism in Mtb and highlights the potential utility of small molecule modulators of cAMP signaling.

Pathway profiling in Mycobacterium tuberculosis: elucidation of cholesterol-derived catabolite and enzymes that catalyze its metabolism.

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-(14)C]cholesterol or [1,7,15,22,26-(13)C]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 1ß-(2'-propanoate)-3aα-H-4α-(3'-propanoic acid)-7aß-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.

Magnesium depletion triggers production of an immune modulating diterpenoid in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is the causative agent of the human disease Tuberculosis, and remains a worldwide health threat responsible for ∼1.7 million deaths annually. During infection, Mtb prevents acidification of the engulfing phagosome, thus blocking endocytic progression and eventually leading to stable residence. The diterpenoid metabolite isotuberculosinol (isoTb) exhibits biological activity indicative of a role in this early arrest of phagosome maturation. Presumably, isoTb production should be induced by phagosomal entry. However, the relevant enzymatic genes are not transcriptionally upregulated during engulfment. Previous examination of the initial biosynthetic enzyme (Rv3377c/MtHPS) involved in isoTb biosynthesis revealed striking inhibition by its Mg(2+) cofactor, leading to the hypothesis that the depletion of Mg(2+) observed upon phagosomal engulfment may act to trigger isoTb biosynthesis. While Mtb is typically grown in relatively high levels of Mg(2+) (0.43 mM), shifting Mtb to media with phagosomal levels (0.1 mM) led to a significant (∼10-fold) increase in accumulation of the MtHPS product, halimadienyl diphosphate, as well as easily detectable amounts of the derived bioactive isoTb. These results demonstrate isoTb production by Mtb specifically under conditions that mimic phagosomal cation concentrations, and further support a role for isoTb in the Mtb infection process.

Iron limitation in M. tuberculosis has broad impact on central carbon metabolism.

Mycobacterium tuberculosis (Mtb), the cause of the human pulmonary disease tuberculosis (TB), contributes to approximately 1.5 million deaths every year. Prior work has established that lipids are actively catabolized by Mtb in vivo and fulfill major roles in Mtb physiology and pathogenesis. We conducted a high-throughput screen to identify inhibitors of Mtb survival in its host macrophage. One of the hit compounds identified in this screen, sAEL057, demonstrates highest activity on Mtb growth in conditions where cholesterol was the primary carbon source. Transcriptional and functional data indicate that sAEL057 limits Mtb's access to iron by acting as an iron chelator. Furthermore, pharmacological and genetic inhibition of iron acquisition results in dysregulation of cholesterol catabolism, revealing a previously unappreciated linkage between these pathways. Characterization of sAEL057's mode of action argues that Mtb's metabolic regulation reveals vulnerabilities in those pathways that impact central carbon metabolism.

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-gamma.

Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated "MKR superspreader", and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR's intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Corrigendum: Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

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Development of a novel, cell-based chemical screen to identify inhibitors of intraphagosomal lipolysis in macrophages.

Macrophages play a central role in tissue homeostasis and the immune system. Their primary function is to internalize cellular debris and microorganisms for degradation within their phagosomes. In this context, their capacity to process and sequester lipids such as triacylglycerides and cholesteryl esters makes them key players in circulatory diseases, such as atheroclerosis. To discover new inhibitors of lipolytic processing within the phagosomal system of the macrophage, we have developed a novel, cell-based assay suitable for high-throughput screening. We employed particles carrying a fluorogenic triglyceride substrate and a calibration fluor to screen for inhibitors of phagosomal lipolysis. A panel of secondary assays were employed to discriminate between lipase inhibitors and compounds that perturbed general phagosomal trafficking events. This process enabled us to identify a new structural class of pyrazole-methanone compounds that directly inhibit lysosomal and lipoprotein lipase activity.

Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D.

Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as alphaVbeta5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.

Immunometabolism at the interface between macrophages and pathogens.

It is generally regarded that the progression of an infection within host macrophages is the consequence of a failed immune response. However, recent appreciation of macrophage heterogeneity, with respect to both development and metabolism, indicates that the reality is more complex. Different lineages of tissue-resident macrophages respond divergently to microbial, environmental and immunological stimuli. The emerging picture that the developmental origin of macrophages determines their responses to immune stimulation and to infection stresses the importance of in vivo infection models. Recent investigations into the metabolism of infecting microorganisms and host macrophages indicate that their metabolic interface can be a major determinant of pathogen growth or containment. This Review focuses on the integration of data from existing studies, the identification of challenges in generating and interpreting data from ongoing studies and a discussion of the technologies and tools that are required to best address future questions in the field.

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.