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SUMMARYDe novolipogenesis (DNL), the process whereby cells synthesize fatty acids from acetyl-coenzyme A (acetyl-CoA), is deregulated in diverse pathologies, including cancer. Here we report that DNL is negatively regulated by Nucleoside Diphosphate Kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently discovered to bind co-enzyme A (CoA). We show that NME1 additionally binds acetyl-CoA and that ligand recognition involves a unique binding mode dependent on the CoA/acetyl-CoA 3’ phosphate. We report thatNme2knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells NME2 mediates a gene transcriptional response to HFD leading to DNL repression and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling acetyl-CoA usage between the competing paths of histone acetylation and DNL.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harbouring key metabolic pathways, including the SUF pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are key for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. We have generated another mutant of the pathway, deficient for the SUFC ATPase, and we have investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on parasite homeostasis. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in growth: cell division and membrane homeostasis are particularly affected. Lipidomic analysis suggests a defect in apicoplast-generated fatty acids, along with a simultaneous increase in scavenging of host-derived lipids. However, addition of exogenous lipids did not allow full restauration of growth, suggesting other more important cellular functions were impacted in addition to fatty acid synthesis. For instance, we have shown that the SUF pathway is also key for generating isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins as well as for the parasite gliding motility. Thus, plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for discovering new potential anti-Toxoplasma targets.

International audience; Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harbouring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are key for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on parasite homeostasis. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis. Our analysis suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P. falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.

Leishmaniasis is a protozoal vector-borne disease that affects both humans and animals. In the Mediterranean Basin, the primary reservoir hosts of Leishmania spp. are mainly rodents and canids. Lipidomic approaches have allowed scientists to establish Leishmania spp. lipid profiles for the identification of cell stage specific biomarkers, drug mechanisms of action, and host immune response. Using an in silico approach of global network interaction between genes involved in fatty acid (FA) synthesis followed by the GC-MS approach, we were able to characterize the fatty acid profiles of L. major derived from human and rodent hosts. Our results revealed that the lipid profile of L. major showed similarities and differences with those already reported for other Leishmania species. Phospholipids are the predominant lipid class. FA composition of rodent parasites was characterized by a lower abundance of the precursor C18:2(n-6). One of the rodent clones, which also expressed the lowest lipid abundance in PL and TAG, was the least sensitive clone to the miltefosine drug and has the lowest infection efficiency. Our findings suggest that the lipid composition variation may explain the response of the parasite toward treatment and their ability to infect their host.

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7., Author summary In this study, we demonstrate that protein kinase TgCDPK7 regulates cellular processes like vesicular trafficking and the synthesis of phospholipids, which are critical for the development of the parasite Toxoplasma gondii. It regulates the localization of a small GTPase TgRab11a by phosphorylating it at a specific site, which is critical for trafficking of important parasite proteins and is important for parasite division. TgCDPK7 may regulate key enzymes involved biogenesis of phosphatidylethanolamine, which may contribute to the synthesis of this important phospholipid. These and other studies shed light on a novel signaling pathway in apicomplexan parasite Toxoplasma gondii.

Host cell invasion and subsequent egress byToxoplasmaparasites is regulated by a network of cGMP, cAMP, and calcium signalling proteins. Such eukaryotic signalling networks typically involve lipid second messengers including phosphatidylinositol phosphates (PIPs), diacylglycerol (DAG) and phosphatidic acid (PA). However, the lipid signalling network inToxoplasmais poorly defined. Here we present lipidomic analysis of a mutant of central flippase/guanylate cyclase TgGC inToxoplasma, which we show has disrupted turnover of signalling lipids impacting phospholipid metabolism and membrane stability. The turnover of signalling lipids is extremely rapid in extracellular parasites and we track changes in PA and DAG to within 5 seconds, which are variably defective upon disruption of TgGC and other signalling proteins. We then identify the position of each protein in the signal chain relative to the central cGMP signalling protein TgGC and map the lipid signal network coordinating conoid extrusion and microneme secretion for egress and invasion.

Summary Human innate immunity to Trypanosoma brucei involves the trypanosome C-terminal kinesin TbKIFC1, which transports internalized trypanolytic factor apolipoprotein L1 (APOL1) within the parasite. We show that TbKIFC1 preferentially associates with cholesterol-containing membranes and is indispensable for mammalian infectivity. Knockdown of TbKIFC1 did not affect trypanosome growth in vitro but rendered the parasites unable to infect mice unless antibody synthesis was compromised. Surface clearance of Variant Surface Glycoprotein (VSG)-antibody complexes was far slower in these cells, which were more susceptible to capture by macrophages. This phenotype was not due to defects in VSG expression or trafficking but to decreased VSG mobility in a less fluid, stiffer surface membrane. This change can be attributed to increased cholesterol level in the surface membrane in TbKIFC1 knockdown cells. Clearance of surface-bound antibodies by T. brucei is therefore essential for infectivity and depends on high membrane fluidity maintained by the cholesterol-trafficking activity of TbKIFC1., Graphical Abstract, Highlights • The TbKIFC1 kinesin preferentially transports cholesterol-containing membranes • TbKIFC1 is absolutely required for parasite growth in mice • Cholesterol trafficking by TbKIFC1 confers high fluidity to the plasma membrane • High membrane fluidity allows antibody clearance necessary for parasite infectivity, Immunology; Microbiology Parasite; Cell Biology

Summary: Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase, TgATS2, shows that the apicoplast provides (lyso)phosphatidic acid, required for the recruitment of a critical dynamin (TgDrpC) during parasite cytokinesis. Disruption of TgATS2 also leads parasites to shift metabolic lipid acquisition from de novo synthesis toward host scavenging. We show that both lipid scavenging and de novo synthesis pathways in wild-type parasites exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (1) upregulation of de novo fatty acid synthesis capacities in the apicoplast and (2) parasite-driven host remodeling to generate multi-membrane-bound structures from host organelles that are imported toward the parasite. : Apicoplast de novo lipid synthesis and lipid host scavenging are both critical for apicomplexan intracellular development. Amiar et al. show that the parasite adapts to the fluctuations of host nutritional content to regulate the metabolic activity of both apicoplast and scavenging pathways and maintain parasite development and division. Keywords: Apicomplexa, toxoplasmosis, malaria, apicoplast, lipid synthesis, phosphatidic acid, host-parasite interaction, lipidomics, host nutritional environment, cytokinesis

Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on intraerythrocytic growth, gametocyte development and gametogenesis. However, parasites lacking PNPLA1 were impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2 . PC synthesis is known to be pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis; we thus hypothesize that the higher PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

Phospholipid metabolism is crucial for membrane dynamics in malaria parasites during entire cycle in the host cell. Plasmodium falciparum harbours several members of phospholipase family, which play key role in phospholipid metabolism. Here we have functionally characterized a parasite lysophospholipase (PfLPL1) with a view to understand its role in lipid homeostasis. We used a regulated fluorescence affinity tagging, which allowed endogenous localization and transient knock-down of the protein. PffLPL1localizes to dynamic vesicular structures that traffic from parasite periphery, through the cytosol to get associated as a multi-vesicular neutral lipid rich body next to the food-vacuole during blood stages. Down-regulation of the PfLPL1 disrupted parasite lipid-homeostasis leading to significant reduction of neutral lipids in lipid-bodies. This hindered conversion of heme to hemozoin, leading to food-vacuole abnormalities, which in turn disrupted parasite development cycle and significantly inhibited parasite growth. Detailed lipidomic analyses of inducible knock-down parasites confirmed role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Our study thus suggests a specific role of PfLPL1 to generate neutral lipids in the parasite, which are essential for parasite survival.ImportancePresent study was undertaken with a view to understand the functional role of a unique lipase (lysophopholipase, PfLPL1) of the human malaria parasite. We utilized genetic approaches for GFP tagging as well as to knock-down the target protein in the parasite. Our studies showed that PfLPL1 associates closely with the lysosome like organelle in the parasite, the food-vacuole. During the blood stage parasite cycle, the food-vacuole is involved in degradation of host haemoglobin and conversion of heme to hemozoin. Genetic knock-down approaches and detailed lipidomic studies confirmed that PfLPL1 protein plays key role in generation of neutral lipid stores in the parasite; neutral lipids are essentially required for hemozoin formation in the parasite, a vital function of the food-vacuole. Overall, this study identified specific role of PfLPL1 in the parasite which is essential for parasite survival.

Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on erythrocytic replication, gametocyte maturation and gametogenesis. However, blood stage parasites lacking PNPLA1 were severely impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2. Because PC synthesis is pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis, we hypothesize that the high PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.

International audience; Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca2+] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca2+ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K+] to activate cytosolic [Ca2+] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca2+ signaling and motility in Toxoplasma.

Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases including malaria and toxoplasmosis. Apicomplexan parasites must obtain and combine lipids both from host cell scavenging andde novosynthesis to maintain parasite propagation and survival within their human host. Major questions on the actual role for each lipid source or how these are regulated upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase TgATS2, shows that the apicoplast provides local (lyso)phosphatidic acid balance, which is required for the recruitment of a novel dynamin (TgDrpC) critical during parasite cytokinesis. Disruption of TgATS2 led parasites to shift metabolic lipid acquisition fromde novosynthesis towards host scavenging. Importantly, both lipid scavenging andde novosynthesis pathways exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (i) up-regulation ofde novofatty acid synthesis capacities in the apicoplast, and (ii) parasite-driven host cell remodelling to generate multi-membrane-bound structures from host organelles that are imported towards the parasite.

We have analyzed the prebiotic effect of softwood hemicelluloses on specific bacterial species in vitro and on microbiota diversity and metabolism in healthy mice in vivo. The ethanol-precipitated glycans from the softwood hemicellulose autohydrolysate were able to stimulate in vitro the growth of Bifidobacterium adolescentis, but to a much lesser extent than that of adherent-invasive E. coli. B. adolescentis was the best producer of SCFA. When mice were fed with the ethanol precipitate fraction, the relative abundance of Bacteroidetes increased while that of Proteobacteria diminished, suggesting change towards a less obesogenic microbiome. Following treatment, lipid analysis showed a decrease in cholesterol, bile acids and free fatty acids, indicating a potential cardioprotection role. Furthermore, analysis of fermentation metabolites revealed an increase in metabolites important for immune health and well-being. Our results highlight the value of hemicelluloses as a potential source of prebiotics supporting both a healthy digestive tract and immune system.

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca(2+)] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca(2+) ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K(+)] to activate cytosolic [Ca(2+)] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca(2+) signaling and motility in Toxoplasma.

Plastids are key organelles in both photosynthetic and nonphotosynthetic organisms. In photosynthetic organisms, plastids can be readily purified using differential centrifugations due to the high density of photosynthetic membranes or thylakoids. The apicomplexan plastid (the apicoplast) is an essential nonphotosynthetic plastid that lacks thylakoid and was not readily purified using conventional methods. Here, we describe a tractable method to purify intact apicoplasts from Plasmodium falciparum blood stages using magnetic beads and affinity purification.

International audience; Chloroplasts are essential organelles that are responsible for photosynthesis in a wide range of organisms that have colonized all biotopes on Earth such as plants and unicellular algae. Interestingly, a secondary endosymbiotic event of a red algal ancestor gave rise to a group of organisms that have adopted an obligate parasitic lifestyle named Apicomplexa parasites. Apicomplexa parasites are some of the most widespread and poorly controlled pathogens in the world. These infectious agents are responsible for major human diseases such as toxoplasmosis, caused by Toxoplasma gondii, and malaria caused by Plasmodium spp. Most of these parasites harbor this relict plastid named the apicoplast, which is essential for parasite survival. The apicoplast has lost photosynthetic capacities but are metabolically similar to plant and algal chloroplasts. The apicoplast is considered a novel and important drug target against Apicomplexa parasites. This chapter focuses on the apicoplast of apicomplexa parasites, its maintenance, and its metabolic pathways.

Chloroplasts are essential organelles that are responsible for photosynthesis in a wide range of organisms that have colonized all biotopes on Earth such as plants and unicellular algae. Interestingly, a secondary endosymbiotic event of a red algal ancestor gave rise to a group of organisms that have adopted an obligate parasitic lifestyle named Apicomplexa parasites. Apicomplexa parasites are some of the most widespread and poorly controlled pathogens in the world. These infectious agents are responsible for major human diseases such as toxoplasmosis, caused by Toxoplasma gondii, and malaria caused by Plasmodium spp. Most of these parasites harbor this relict plastid named the apicoplast, which is essential for parasite survival. The apicoplast has lost photosynthetic capacities but are metabolically similar to plant and algal chloroplasts. The apicoplast is considered a novel and important drug target against Apicomplexa parasites. This chapter focuses on the apicoplast of apicomplexa parasites, its maintenance, and its metabolic pathways.

Plastids are key organelles in both photosynthetic and nonphotosynthetic organisms. In photosynthetic organisms, plastids can be readily purified using differential centrifugations due to the high density of photosynthetic membranes or thylakoids. The apicomplexan plastid (the apicoplast) is an essential nonphotosynthetic plastid that lacks thylakoid and was not readily purified using conventional methods. Here, we describe a tractable method to purify intact apicoplasts from Plasmodium falciparum blood stages using magnetic beads and affinity purification.

Pathogenic species of Mycobacteria and Corynebacteria, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, synthesize complex cell walls that are rich in very long-chain mycolic acids. These fatty acids are synthesized on the inner leaflet of the cell membrane and are subsequently transported to the periplasmic space as trehalose monomycolates (TMM), where they are conjugated to other cell wall components and to TMM to form trehalose dimycolates (TDM). Mycobacterial TMM, and the equivalent Corynebacterium glutamicum trehalose corynomycolates (TMCM), are transported across the inner membrane by MmpL3, or NCgl0228 and NCgl2769, respectively, although little is known about how this process is regulated. Here, we show that transient acetylation of the mycolyl moiety of TMCM is required for periplasmic export. A bioinformatic search identified a gene in a cell wall biosynthesis locus encoding a putative acetyltransferase (M. tuberculosis Rv0228/C. glutamicum NCgl2759) that was highly conserved in all sequenced Corynebacterineae. Deletion of C. glutamicum NCgl2759 resulted in the accumulation of TMCM, with a concomitant reduction in surface transport of this glycolipid and syntheses of cell wall trehalose dicorynomycolates. Strikingly, loss of NCgl2759 was associated with a defect in the synthesis of a minor, and previously uncharacterized, glycolipid species. This lipid was identified as trehalose monoacetylcorynomycolate (AcTMCM) by mass spectrometry and chemical synthesis of the authentic standard. The in vitro synthesis of AcTMCM was dependent on acetyl-CoA, whereas in vivo [(14)C]-acetate pulse-chase labeling showed that this lipid was rapidly synthesized and turned over in wild-type and genetically complemented bacterial strains. Significantly, the biochemical and TMCM/TDCM transport phenotype observed in the ΔNCgl2759 mutant was phenocopied by inhibition of the activities of the two C. glutamicum MmpL3 homologues. Collectively, these data suggest that NCgl2759 is a novel TMCM mycolyl acetyltransferase (TmaT) that regulates transport of TMCM and is a potential drug target in pathogenic species.

International audience; Patatin-like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin-like catalytic domain, whose phospholipase activity relies on a serine-aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin-like phospholipase domain-containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin-like domain. Using immunofluorescence assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast-generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite.

Mycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760 t-LM comprised a series of short LM species (8–27 residues long) containing an α1–6-linked mannose backbone with greatly reduced α1–2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane α1–6-mannosyltransferase involved in extending the α1–6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

Phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) are essential components of the cell wall and plasma membrane of mycobacteria, including the human pathogen Mycobacterium tuberculosis, as well as the related Corynebacterineae. We have previously shown that the lipoprotein, LpqW, regulates PIM and LM/LAM biosynthesis in mycobacteria. Here, we provide direct evidence that LpqW regulates the activity of key mannosyltransferases in the periplasmic leaflet of the cell membrane. Inactivation of the Corynebacterium glutamicum lpqW ortholog, NCgl1054, resulted in a slow growth phenotype and a global defect in lipoglycan biosynthesis. The NCgl1054 mutant lacked LAMs and was defective in the elongation of the major PIM species, AcPIM2, as well as a second glycolipid, termed Gl-X (mannose-α1-4-glucuronic acid-α1-diacylglycerol), which function as membrane anchors for LM-A and LM-B, respectively. Elongation of AcPIM2 and Gl-X was found to be dependent on expression of polyprenol phosphomannose (ppMan) synthase. However, the ΔNCgl1054 mutant synthesized normal levels of ppMan, indicating that LpqW is not required for synthesis of this donor. A spontaneous suppressor strain was isolated in which lipoglycan synthesis in the ΔNCgl1054 mutant was partially restored. Genome-wide sequencing indicated that a single amino acid substitution within the ppMan-dependent mannosyltransferase MptB could bypass the need for LpqW. Further evidence of an interaction is provided by the observation that MptB activity in cell-free extracts was significantly reduced in the absence of LpqW. Collectively, our results suggest that LpqW may directly activate MptB, highlighting the role of lipoproteins in regulating key cell wall biosynthetic pathways in these bacteria.

International audience; Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main lipids in photosynthetic membranes in plant cells. They are synthesized in the envelope surrounding plastids by MGD and DGD galactosyltransferases. These galactolipids are critical for the biogenesis of photosynthetic membranes, and they act as a source of polyunsaturated fatty acids for the whole cell and as phospholipid surrogates in phosphate shortage. Based on a high-throughput chemical screen, we have characterized a new compound, galvestine-1, that inhibits MGDs in vitro by competing with diacylglycerol binding. Consistent effects of galvestine-1 on Arabidopsis thaliana include root uptake, circulation in the xylem and mesophyll, inhibition of MGDs in vivo causing a reduction of MGDG content and impairment of chloroplast development. The effects on pollen germination shed light on the contribution of galactolipids to pollen-tube elongation. The whole-genome transcriptional response of Arabidopsis points to the potential benefits of galvestine-1 as a unique tool to study lipid homeostasis in plants.

The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature.

Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion., SCOPUS: ar.j, info:eu-repo/semantics/published

The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii, extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide-phospholipase C (PLC) activation, which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product, phosphatidic acid (PA), is essential for controlling microneme release. Governing this balance is the apicomplexan-specific DAG-kinase-1, which interconverts PA and DAG, and whose depletion impairs egress and causes parasite death. Additionally, we identify an acylated pleckstrin-homology (PH) domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa, these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis.

The human malaria parasite Plasmodium falciparum harbors a relict, nonphotosynthetic plastid of algal origin termed the apicoplast. Although considerable progress has been made in defining the metabolic functions of the apicoplast, information on the composition and biogenesis of the four delimiting membranes of this organelle is limited. Here, we report an efficient method for preparing highly purified apicoplasts from red blood cell parasite stages and the comprehensive lipidomic analysis of this organelle. Apicoplasts were prepared from transgenic parasites expressing an epitope-tagged triosephosphate transporter and immunopurified on magnetic beads. Gas and liquid chromatography MS analyses of isolated apicoplast lipids indicated significant differences compared with total parasite lipids. In particular, apicoplasts were highly enriched in phosphatidylinositol, consistent with a suggested role for phosphoinositides in targeting membrane vesicles to apicoplasts. Apicoplast phosphatidylinositol and other phospholipids were also enriched in saturated fatty acids, which could reflect limited acyl exchange with other membrane phospholipids and/or a requirement for specific physical properties. Lipids atypical for plastids (sphingomyelins, ceramides, and cholesterol) were detected in apicoplasts. The presence of cholesterol in apicoplast membranes was supported by filipin staining of isolated apicoplasts. Galactoglycerolipids, dominant in plant and algal plastids, were not detected in P. falciparum apicoplasts, suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle. Apicoplasts thus contain an atypical melange of lipids scavenged from the human host alongside lipids remodeled by the parasite cytoplasm, and stable isotope labeling shows some apicoplast lipids are generated de novo by the organelle itself.

International audience; Plant cells are characterized by the presence of chloroplasts, membrane lipids of which contain up to ∼80% mono- and digalactosyldiacylglycerol (MGDG and DGDG). The synthesis of MGDG in the chloroplast envelope is essential for the biogenesis and function of photosynthetic membranes, is coordinated with lipid metabolism in other cell compartments and is regulated in response to environmental factors. Phenotypic analyses of Arabidopsis using the recently developed specific inhibitor called galvestine-1 complete previous analyses performed using various approaches, from enzymology, cell biology to genetics. This review details how this probe could be beneficial to study the lipid homeostasis system at the whole cell level and highlights connections between MGDG synthesis and Arabidopsis flower development.