Metabolic stringent response in intracellular stages of Leishmania.

Leishmania are unusual in being able to survive long-term in the mature phagolysosome compartment of macrophages and other phagocytic cells in their mammalian hosts. Key to their survival in this niche, Leishmania amastigotes switch to a slow growth state and activate a stringent metabolic response. The stringent metabolic response may be triggered by multiple stresses and is associated with decreased metabolic fluxes, restricted use of sugars and fatty acids as carbon sources and increased dependence on metabolic homeostasis pathways. Heterogeneity in expression of the Leishmania stringent response occurs in vivo reflects temporal and spatial heterogeneity in lesion tissues and includes non-dividing dormant stages. This response underpins the capacity of these parasites to maintain long-term chronic infections and survive drug treatments.

Evolution and function of carbohydrate reserve biosynthesis in parasitic protists.

Nearly all eukaryotic cells synthesize carbohydrate reserves, such as glycogen, starch, or low-molecular-weight oligosaccharides. However, a number of parasitic protists have lost this capacity while others have lost, and subsequently evolved, entirely new pathways. Recent studies suggest that retention, loss, or acquisition of these pathways in different protists is intimately linked to their lifestyle. In particular, parasites with carbohydrate reserves often establish long-lived chronic infections and/or produce environmental cysts, whereas loss of these pathways is associated with parasites that have highly proliferative and metabolically active life-cycle stages. The evolution of mannogen biosynthesis in Leishmania and related parasites indicates that these pathways have played a role in defining the host range and niches occupied by some protists.

A Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites.

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of ß-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.

The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell.

Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In Toxoplasma gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here, we demonstrate that the ER-localized fatty acid elongation (ELO) complexes are essential for parasite growth. Conditional knockdown of the nonredundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. (13) C-glucose and (13) C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long-chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was bypassed by supplementing the media with specific fatty acids, indicating active but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

Acetylation of trehalose mycolates is required for efficient MmpL-mediated membrane transport in Corynebacterineae.

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.

Golgi-located NTPDase1 of Leishmania major is required for lipophosphoglycan elongation and normal lesion development whereas secreted NTPDase2 is dispensable for virulence.

Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.

Leishmania major methionine sulfoxide reductase A is required for resistance to oxidative stress and efficient replication in macrophages.

Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H(2)O(2) compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H(2)O(2) and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.

Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.

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.

Discovery of inhibitors of Leishmania ß-1,2-mannosyltransferases using a click-chemistry-derived guanosine monophosphate library.

Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of ß-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose-dependent ß-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-D-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen ß-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism.

Central carbon metabolism of Leishmania parasites.

Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.

Use of click chemistry to define the substrate specificity of Leishmania beta-1,2-mannosyltransferases.

Leishmania spp. are human pathogens that utilize a novel beta-1,2-mannan as their major carbohydrate reserve material. We describe a new approach that combines traditional substrate-modification methods and "click chemistry" to assemble a library of modified substrates that were used to qualitatively define the substrate tolerance of the Leishmania beta-1,2-mannosyltransferases responsible for beta-1,2-mannan biosynthesis. The library was assembled by using the highly selective copper(I)-catalysed cycloaddition reaction of azides and alkynes to couple an assortment of azide- and alkyne-functionalized small molecules with complementary alkyne- and azide-functionalized mannose derivatives. All mannose derivatives with alpha-orientated substituents on the anomeric carbon were found to act as substrates when incubated with a Leishmania mexicana particulate fraction containing GDP-mannose. In contrast, 6-substituted mannose derivatives were not substrates. Representative products formed from the library compounds were analysed by mass spectrometry, methylation linkage analysis and beta-mannosidase digestions and showed extension with up to four beta-1,2-linked mannosyl residues. This work provides insights into the substrate specificity of this new class of glycosyltransferases that can be applied to the development of highly specific tools and inhibitors for their study.

Leishmania beta-1,2-mannan is assembled on a mannose-cyclic phosphate primer.

Infective stages of the protozoan parasite Leishmania spp. accumulate a class of beta-1,2-mannan oligosaccharides as their major carbohydrate reserve material. Here, we describe the biosynthesis of Leishmania mannan. Mannan precursors were identified by metabolic labeling of Leishmania mexicana promastigotes with [(3)H]mannose. Label was initially incorporated into a phosphomannose primer and short phosphorylated beta-1,2-mannan oligomers that were two to five residues long. Analysis of the mannan primer by Fourier transform ion-cyclotron resonance MS and various enzymatic and chemical treatments and comparison with authentic mannose (Man) phosphates indicated the presence of Man-alpha-1,4-cyclic phosphate. This primer was synthesized from Man-6-phosphate by means of Man-1-phosphate in a cell-free system. Short mannan chains containing the primer were subsequently dephosphorylated and then further elongated by GDP-Man-dependent transferases in vivo and in the cell-free system. The synthesis of this glycan primer likely constitutes a key regulatory step in mannan biosynthesis and is a potential target for antileishmanial drugs.

Evidence that intracellular beta1-2 mannan is a virulence factor in Leishmania parasites.

The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.

Intracellular trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins and free GPIs in Leishmania mexicana.

Free glycosylphosphatidylinositols (GPIs) are an important class of membrane lipids in many pathogenic protozoa. In this study, we have investigated the subcellular distribution and intracellular trafficking of an abundant class of free GPIs [termed glycosylinositolphospholipids (GIPLs)] in Leishmania mexicana promastigotes. The intracellular transport of the GIPLs and the major GPI-anchored glycoprotein gp63 was measured by following the incorporation of these molecules into sphingolipid-rich, detergent-resistant membranes (DRMs) in the plasma membrane. In metabolic-labelling experiments, mature GIPLs and gp63 were transported to DRMs in the plasma membrane with a t(1/2) of 70 and 40 min, respectively. Probably, GIPL transport to the DRMs involves a vesicular mechanism, as transport of both the GIPLs and gp63 was inhibited similarly at 10 degrees C. All GIPL intermediates were quantitatively recovered in Triton X-100-soluble membranes and were largely orientated on the cytoplasmic face of the endoplasmic reticulum, as shown by their sensitivity to exogenous phosphatidylinositol-specific phospho-lipase C. On the contrary, a significant proportion of the mature GIPLs ( approximately 50% of iM4) were accessible to membrane-impermeable probes on the surface of live promastigotes. These results suggest that the GIPLs are flipped across intracellular or plasma membranes during surface transport and that a significant fraction may populate the cytoplasmic leaflet of the plasma membrane. Finally, treatment of L. mexicana promastigotes with myriocin, an inhibitor of sphingolipid biosynthesis, demonstrated that ongoing sphingolipid biosynthesis is not required for the plasma-membrane transport of either gp63 or the GIPLs and that DRMs persist even when cellular levels of the major sphingolipid are depleted by 70%.