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.

Mycobacterium tuberculosis requires glyoxylate shunt and reverse methylcitrate cycle for lactate and pyruvate metabolism.

Bacterial nutrition is an essential aspect of host-pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a 'multi-omics' approach consisting of transposon-directed insertion site sequencing (TraDIS), RNA-seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry-based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl-CoA, the essential precursor for the biosynthesis of the odd-chain fatty acids.

Matrix stiffening promotes a tumor vasculature phenotype.

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.

Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling.

Mycobacterium tuberculosis (Mtb) uses a complex 3', 5'-cyclic AMP (cAMP) signaling network to sense and respond to changing environments encountered during infection, so perturbation of cAMP signaling might be leveraged to disrupt Mtb pathogenesis. However, understanding of cAMP signaling pathways is hindered by the presence of at least 15 distinct adenylyl cyclases (ACs). Recently, the small molecule V-58 was shown to inhibit Mtb replication within macrophages and stimulate cAMP production in Mtb. Here we determined that V-58 rapidly and directly activates Mtb AC Rv1625c to produce high levels of cAMP regardless of the bacterial environment or growth medium. Metabolic inhibition by V-58 was carbon source dependent in Mtb and did not occur in Mycobacterium smegmatis, suggesting that V-58-mediated growth inhibition is due to interference with specific Mtb metabolic pathways rather than a generalized cAMP toxicity. Chemical stimulation of cAMP production by Mtb within macrophages also caused down regulation of TNF-α production by the macrophages, indicating a complex role for cAMP in Mtb pathogenesis. Together these studies describe a novel approach for targeted stimulation of cAMP production in Mtb, and provide new insights into the myriad roles of cAMP signaling in Mtb, particularly during Mtb's interactions with macrophages.

Thrombospondin-1-deficient mice are not protected from bleomycin-induced pulmonary fibrosis.

Thrombospondin-1 (TSP-1) is an extracellular protein critical to normal lung homeostasis, and is reported to activate latent transforming growth factor-ß (TGF-ß). Because active TGF-ß is causally involved in lung fibrosis after bleomycin challenge, alterations in TSP-1 may be relevant to pulmonary fibrosis. We sought to determine the effects of TSP-1 deficiency on the susceptibility to bleomycin-induced pulmonary fibrosis in a murine model. Age-matched and sex-matched C57BL/6 wild-type (WT) and TSP-1-deficient mice were treated twice weekly for 4 weeks with intraperitoneal bleomycin (0.035 U/g) or PBS, and were allowed to rest 1 week before being killed. Their lungs were inflated with PBS, fixed in formalin, paraffin-embedded, and sectioned. A certified veterinary pathologist blindly scored each slide for inflammation and fibrosis. Lungs were homogenized to obtain RNA and protein for the real-time RT-PCR analysis of connective tissue growth factor (CTGF) and collagen I, and for Western blotting to detect phospho-Smad2, or total Smad2/3, respectively. In response to bleomycin treatment, measures of fibrosis and inflammation, along with CTGF and collagen I mRNA concentrations, were increased in TSP-1-deficient mice compared with WT mice. Notably, Smad 2/3 signaling was of equal strength in WT and TSP-1 knockout mice treated with bleomycin, suggesting that TSP-1 is not required for the activation of TGF-ß. These results demonstrate that TSP-1 deficiency does not protect mice from systemic bleomycin challenge, and that TSP-1 deficiency is associated with increased expression of lung collagen and CTGF.

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.

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.

Activation of estrogen receptor-alpha reduces aortic smooth muscle differentiation.

Women are at high risk of dying from unrecognized cardiovascular disease. Many differences in cardiovascular disease between men and women appear to be mediated by vascular smooth muscle cells (SMC). Because estrogen reduces the proliferation of SMC, we hypothesized that activation of estrogen receptor-alpha (ERalpha) by agonists or by growth factors altered SMC function. To determine the effect of growth factors, estrogen, and ERalpha expression on SMC differentiation, human aortic SMC were cultured in serum-free conditions for 10 days. SMC from men had lower spontaneous expression of ERalpha and higher levels of the differentiation markers calponin and smooth muscle alpha-actin than SMC from women. When SMC containing low expression of ERalpha were transduced with a lentivirus containing ERalpha, activation of the receptor by ligands or growth factors reduced differentiation markers. Conversely, inhibiting ERalpha expression by small interfering RNA (siRNA) in cells expressing high levels of ERalpha enhanced the expression of differentiation markers. ERalpha expression and activation reduced the phosphorylation of Smad2, a signaling molecule important in differentiation of SMC and initiated cell death through cleavage of caspase-3. We conclude that ERalpha activation switched SMC to a dedifferentiated phenotype and may contribute to plaque instability.

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.

Estradiol increases platelet aggregation in Pl(A1/A1) individuals.

BACKGROUND: The platelet glycoprotein IIb/IIIa receptor is a key mediator of platelet aggregation and intracoronary thrombosis. Studies have suggested that hormone replacement therapy (HRT) may increase coronary events in postmenopausal women. OBJECTIVES: We sought to characterize the relationship between the estrogen concentration expected in HRT and platelet aggregation. DESIGN AND RESULTS: Platelet aggregation studies were performed using epinephrine on 30 healthy individuals (15 Pl(A1/A1) and 15 Pl(A1/A2)) before and after incubation with beta-estradiol (E2) (10(-11) mol/L). The effect of E2 10(-11) mol/L on Pl(A1/A1) platelets demonstrated a significant increase (P = .03) in aggregation compared with baseline. In contrast, with the same concentration of E2, aggregation of Pl(A1/A2) platelets decreased significantly compared with baseline (P < .0001). CONCLUSIONS: Estrogen concentration similar to that expected in HRT resulted in an increase in platelet aggregation in Pl(A1/A1) individuals, but not in Pl(A1/A2) individuals. The data may provide further insight for the increase in coronary events seen in HRT clinical trials and suggest that further evaluation is needed to better define the role of pharmacogenetics in HRT.

Screen for small molecules increasing the mitochondrial membrane potential.

The identification of small molecules that positively modulate the mitochondrial respiratory function has broad applications in fundamental research, therapeutic target validation, and drug discovery. We present an approach in which primary screens for mitochondrial function in yeast are used to efficiently identify a subset of high-value compounds that can in turn be rapidly tested against a broad range of mammalian cell lines. The ability of the yeast assay to successfully identify in a high-throughput format hit compounds that increase the mitochondrial membrane potential and adenosine triphosphate (ATP) levels by as little as 15% was demonstrated. In this study, 14 hits were identified from a collection of 13,680 compounds. Secondary testing with myotubes, fibroblasts, and PC-12 and HepG2 cells identified two compounds increasing ATP levels in hepatocytes and two other compounds increasing ATP in fibroblasts. The effect on hepatocytes was further studied using genomic and mitochondrial proteomic tools to characterize the changes induced by the two compounds. Changes in the accumulation of a series of factors involved in early gene response or apoptosis or linked to metabolic functions (i.e., ß-Klotho, RORα, PGC-1α, G6PC, IGFBP1, FTL) were discovered.

Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration.

Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability--a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or small interfering RNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.

NAC and DTT promote TGF-beta1 monomer formation: demonstration of competitive binding.

TGF-Beta plays an important role in the genesis and progression of pulmonary fibrosis. We sought to determine the role of mononuclear phagocytes in the activation of TGF-beta and found that freshly isolated peripheral blood monocytes spontaneously released TGF-beta. Stimulating these monocytes with GM-CSF or LPS, but not MCSF, augmented the activation of TGF-beta. In human monocytes, the free thiol compounds DTT and NAC decreased the activity of TGF-beta, without affecting TGF-beta mRNA transcription. Both NAC and DTT lessened the biological activity of recombinant active TGF-beta in a cell-free system. We found that NAC and DTT reduced dimeric active TGF-beta from a 25 kDa protein to 12.5 kDa inactive monomer. This conversion was reversed using the oxidizing agent diamide. Diamide also restored biological activity to NAC or DTT-treated TGF-beta. Reduction of TGF-beta to monomers could competitively inhibit active dimeric TGF-beta and block intracellular signaling events. Our observations suggest that modulation of the oxidative state of TGF-beta may be a novel therapeutic approach for patients with pulmonary fibrosis.

M-CSF induces vascular endothelial growth factor production and angiogenic activity from human monocytes.

The impact of the immune response in malignancy is poorly understood. While immune cells can destroy transformed cells, the targeting and accumulation of monocytes and macrophages at tumor sites may promote tumor metastases. The growth factor M-CSF is important in promoting monocyte survival. Since M-CSF(-/-) mice are protected against tumor metastases, we hypothesized that M-CSF induced monocytes to produce angiogenic factors that facilitate metastases. In this study we demonstrate that recombinant human M-CSF induces freshly isolated normal human monocytes to produce and release the growth factor vascular endothelial growth factor (VEGF) in a dose-dependent manner, which peaked at 5 days in culture. VEGF released by these monocytes is biologically active, as cell-free supernatants from these M-CSF-stimulated monocytes induced tube formation in HUVEC. Network formation by these HUVECs after treatment with supernatants from monocytes stimulated with M-CSF were inhibited by anti-VEGF, but not by the isogenic control, Abs. Collectively, these data support an important role for M-CSF and monocytes in VEGF production and angiogenesis.

Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells.

This study analyzed the regulation of alpha2-adrenoceptors (alpha2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of alpha2-ARs (alpha2C > alpha2A, via RNase protection assay) and responded to alpha2-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 microM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express alpha2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although alpha2-ARs (alpha2C >> alpha2A) were detected in aortas, alpha2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to alpha2-AR stimulation. Reporter constructs demonstrated higher activities for alpha2A- and alpha2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of alpha2C-AR mRNA and protein but decreased expression of alpha2A-ARs. Serum induction of alpha2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 microM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 microM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the alpha2A-AR antagonist BRL-44408 (100 nM) but not by the alpha2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of alpha2-ARs in human VSMs that reflects differential activity of alpha2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that alpha2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of alpha2C-ARs may contribute to pathological changes in vascular function.