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.

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.

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.

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.

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.

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

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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.

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.

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.

The genetic requirements of fatty acid import by Mycobacterium tuberculosis within macrophages.

Mycobacterium tuberculosis (Mtb) imports and metabolizes fatty acids to maintain infection within human macrophages. Although this is a well-established paradigm, the bacterial factors required for fatty acid import are poorly understood. Previously, we found that LucA and Mce1 are required for fatty acid import in Mtb (Nazarova et al., 2017). Here, we identified additional Mtb mutants that have a reduced ability to import a fluorescent fatty acid substrate during infection within macrophages. This screen identified the novel genes as rv2799 and rv0966c as be necessary for fatty acid import and confirmed the central role for Rv3723/LucA and putative components of the Mce1 fatty acid transporter (Rv0200/OmamB, Rv0172/Mce1D, and Rv0655/MceG) in this process.

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.

Cholesterol and fatty acids grease the wheels of Mycobacterium tuberculosis pathogenesis.

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.

Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we determined that this mutant assimilated 10-fold less Bodipy-palmitate than the wild type strain during infection in macrophages. This quantitative method of fatty acid uptake can be used to further identify pathways involved in lipid uptake by intracellular Mtb and possibly other bacteria.

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.

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.

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 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.

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

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.

The Minimal Unit of Infection: Mycobacterium tuberculosis in the Macrophage.

The interaction between Mycobacterium tuberculosis and its host cell is highly complex and extremely intimate. Were it not for the disease, one might regard this interaction at the cellular level as an almost symbiotic one. The metabolic activity and physiology of both cells are shaped by this coexistence. We believe that where this appreciation has greatest significance is in the field of drug discovery. Evolution rewards efficiency, and recent data from many groups discussed in this review indicate that M. tuberculosis has evolved to utilize the environmental cues within its host to control large genetic programs or regulons. But these regulons may represent chinks in the bacterium's armor because they include off-target effects, such as the constraint of the metabolic plasticity of M. tuberculosis. A prime example is how the presence of cholesterol within the host cell appears to limit the ability of M. tuberculosis to fully utilize or assimilate other carbon sources. And that is the reason for the title of this review. We believe firmly that, to understand the physiology of M. tuberculosis and to identify new drug targets, it is imperative that the bacterium be interrogated within the context of its host cell. The constraints induced by the environmental cues present within the host cell need to be preserved and exploited. The M. tuberculosis-infected macrophage truly is the "minimal unit of infection."