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Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival. [ABSTRACT FROM AUTHOR]

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30–40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial masse increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast. [ABSTRACT FROM AUTHOR]

Fatty acids affect a number of physiological processes, in addition to forming the building blocks of membranes and body fat stores. In this study, we uncover a role for the monounsaturated fatty acid oleate in the innate immune response of the nematode Caenorhabditis elegans. From an RNAi screen for regulators of innate immune defense genes, we identified the two stearoyl-coenzyme A desaturases that synthesize oleate in C. elegans. We show that the synthesis of oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Together, these data add to the known health-promoting effects of monounsaturated fatty acids, and suggest an ancient link between nutrient stores, metabolism, and host susceptibility to bacterial infection. [ABSTRACT FROM AUTHOR]

Combination antiretroviral therapy (cART) dramatically improves survival of HIV-infected patients, but lifelong treatment can ultimately result in cumulative toxicities and drug resistance, thus necessitating the development of new drugs with significantly improved pharmaceutical profiles. We recently found that the fusion inhibitor T-20 (enfuvirtide)-based lipopeptides possess dramatically increased anti-HIV activity. Herein, a group of novel lipopeptides were designed with different lengths of fatty acids, identifying a stearic acid-modified lipopeptide (LP-80) with the most potent anti-HIV activity. It inhibited a large panel of divergent HIV subtypes with a mean IC50 in the extremely low picomolar range, being > 5,300-fold more active than T-20 and the neutralizing antibody VRC01. It also sustained the potent activity against T-20-resistant mutants and exhibited very high therapeutic selectivity index. Pharmacokinetics of LP-80 in rats and monkeys verified its potent and long-acting anti-HIV activity. In the monkey, subcutaneous administration of 3 mg/kg LP-80 yielded serum concentrations of 1,147 ng/ml after injection 72 h and 9 ng/ml after injection 168 h (7 days), equivalent to 42,062- and 330-fold higher than the measured IC50 value. In SHIV infected rhesus macaques, a single low-dose LP-80 (3 mg/kg) sharply reduced viral loads to below the limitation of detection, and twice-weekly monotherapy could maintain long-term viral suppression. [ABSTRACT FROM AUTHOR]

Positive strand RNA viruses, such as dengue virus type 2 (DENV2) expand and structurally alter ER membranes to optimize cellular communication pathways that promote viral replicative needs. These complex rearrangements require significant protein scaffolding as well as changes to the ER chemical composition to support these structures. We have previously shown that the lipid abundance and repertoire of host cells are significantly altered during infection with these viruses. Specifically, enzymes in the lipid biosynthesis pathway such as fatty acid synthase (FAS) are recruited to viral replication sites by interaction with viral proteins and displayed enhanced activities during infection. We have now identified that events downstream of FAS (fatty acid desaturation) are critical for virus replication. In this study we screened enzymes in the unsaturated fatty acid (UFA) biosynthetic pathway and found that the rate-limiting enzyme in monounsaturated fatty acid biosynthesis, stearoyl-CoA desaturase 1 (SCD1), is indispensable for DENV2 replication. The enzymatic activity of SCD1, was required for viral genome replication and particle release, and it was regulated in a time-dependent manner with a stringent requirement early during viral infection. As infection progressed, SCD1 protein expression levels were inversely correlated with the concentration of viral dsRNA in the cell. This modulation of SCD1, coinciding with the stage of viral replication, highlighted its function as a trigger of early infection and an enzyme that controlled alternate lipid requirements during early versus advanced infections. Loss of function of this enzyme disrupted structural alterations of assembled viral particles rendering them non-infectious and immature and defective in viral entry. This study identifies the complex involvement of SCD1 in DENV2 infection and demonstrates that these viruses alter ER lipid composition to increase infectivity of the virus particles. [ABSTRACT FROM AUTHOR]

De novo biosynthesis of lipids is essential for Trypanosoma brucei, a protist responsible for the sleeping sickness. Here, we demonstrate that the ketogenic carbon sources, threonine, acetate and glucose, are precursors for both fatty acid and sterol synthesis, while leucine only contributes to sterol production in the tsetse fly midgut stage of the parasite. Degradation of these carbon sources into lipids was investigated using a combination of reverse genetics and analysis of radio-labelled precursors incorporation into lipids. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway. [ABSTRACT FROM AUTHOR]

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes. [ABSTRACT FROM AUTHOR]

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease. [ABSTRACT FROM AUTHOR]

Intracellular infection and multi-organ colonization by the protozoan parasite, Trypanosoma cruzi, underlie the complex etiology of human Chagas disease. While T. cruzi can establish cytosolic residence in a broad range of mammalian cell types, the molecular mechanisms governing this process remain poorly understood. Despite the anticipated capacity for fatty acid synthesis in this parasite, recent observations suggest that intracellular T. cruzi amastigotes may rely on host fatty acid metabolism to support infection. To investigate this prediction, it was necessary to establish baseline lipidome information for the mammalian-infective stages of T. cruzi and their mammalian host cells. An unbiased, quantitative mass spectrometric analysis of lipid fractions was performed with the identification of 1079 lipids within 30 classes. From these profiles we deduced that T. cruzi amastigotes maintain an overall lipid identity that is distinguishable from mammalian host cells. A deeper analysis of the fatty acid moiety distributions within each lipid subclass facilitated the high confidence assignment of host- and parasite-like lipid signatures. This analysis unexpectedly revealed a strong host lipid signature in the parasite lipidome, most notably within its glycerolipid fraction. The near complete overlap of fatty acid moiety distributions observed for host and parasite triacylglycerols suggested that T. cruzi amastigotes acquired a significant portion of their lipidome from host triacylglycerol pools. Metabolic tracer studies confirmed long-chain fatty acid scavenging by intracellular T. cruzi amastigotes, a capacity that was significantly diminished in host cells deficient for de novo triacylglycerol synthesis via the diacylglycerol acyltransferases (DGAT1/2). Reduced T. cruzi amastigote proliferation in DGAT1/2-deficient fibroblasts further underscored the importance of parasite coupling to host triacylglycerol pools during the intracellular infection cycle. Thus, our comprehensive lipidomic dataset provides a substantially enhanced view of T. cruzi infection biology highlighting the interplay between host and parasite lipid metabolism with potential bearing on future therapeutic intervention strategies. [ABSTRACT FROM AUTHOR]

Toxoplasma is an obligate intracellular parasite that replicates in mammalian cells within a parasitophorous vacuole (PV) that does not fuse with any host organelles. One mechanism developed by the parasite for nutrient acquisition is the attraction of host organelles to the PV. Here, we examined the exploitation of host lipid droplets (LD), ubiquitous fat storage organelles, by Toxoplasma. We show that Toxoplasma replication is reduced in host cells that are depleted of LD, or impaired in TAG lipolysis or fatty acid catabolism. In infected cells, the number of host LD and the expression of host LD-associated genes (ADRP, DGAT2), progressively increase until the onset of parasite replication. Throughout infection, the PV are surrounded by host LD. Toxoplasma is capable of accessing lipids stored in host LD and incorporates these lipids into its own membranes and LD. Exogenous addition of oleic acid stimulates LD biogenesis in the host cell and results in the overaccumulation of neutral lipids in very large LD inside the parasite. To access LD-derived lipids, Toxoplasma intercepts and internalizes within the PV host LD, some of which remaining associated with Rab7, which become wrapped by an intravacuolar network of membranes (IVN). Mutant parasites impaired in IVN formation display diminished capacity of lipid uptake from host LD. Moreover, parasites lacking an IVN-localized phospholipase A2 are less proficient in salvaging lipids from host LD in the PV, suggesting a major contribution of the IVN for host LD processing in the PV and, thus lipid content release. Interestingly, gavage of parasites with lipids unveils, for the first time, the presence in Toxoplasma of endocytic-like structures containing lipidic material originating from the PV lumen. This study highlights the reliance of Toxoplasma on host LD for its intracellular development and the parasite’s capability in scavenging neutral lipids from host LD. [ABSTRACT FROM AUTHOR]

Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication. [ABSTRACT FROM AUTHOR]

During a tuberculosis infection and inside lipid-laden foamy macrophages, fatty acids (FAs) and sterols are the major energy and carbon source for Mycobacterium tuberculosis. Mycobacteria can be found both inside a vacuole and the cytosol, but how this impacts their access to lipids is not well appreciated. Lipid droplets (LDs) store FAs in form of triacylglycerols (TAGs) and are energy reservoirs of prokaryotes and eukaryotes. Using the Dictyostelium discoideum/Mycobacterium marinum infection model we showed that M. marinum accesses host LDs to build up its own intracytosolic lipid inclusions (ILIs). Here, we show that host LDs aggregate at regions of the bacteria that become exposed to the cytosol, and appear to coalesce on their hydrophobic surface leading to a transfer of diacylglycerol O-acyltransferase 2 (Dgat2)-GFP onto the bacteria. Dictyostelium knockout mutants for both Dgat enzymes are unable to generate LDs. Instead, the excess of exogenous FAs is esterified predominantly into phospholipids, inducing uncontrolled proliferation of the endoplasmic reticulum (ER). Strikingly, in absence of host LDs, M. marinum alternatively exploits these phospholipids, resulting in rapid reversal of ER-proliferation. In addition, the bacteria are unable to restrict their acquisition of lipids from the dgat1&2 double knockout leading to vast accumulation of ILIs. Recent data indicate that the presence of ILIs is one of the characteristics of dormant mycobacteria. During Dictyostelium infection, ILI formation in M. marinum is not accompanied by a significant change in intracellular growth and a reduction in metabolic activity, thus providing evidence that storage of neutral lipids does not necessarily induce dormancy. [ABSTRACT FROM AUTHOR]

Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1–83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1–83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1–240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88. [ABSTRACT FROM AUTHOR]

Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii. [ABSTRACT FROM AUTHOR]