Proteins involved in the biosynthesis of lipophosphoglycan in Leishmania: a comparative genomic and evolutionary analysis.

BACKGROUND: Leishmania spp. are digenetic parasites capable of infecting humans and causing a range of diseases collectively known as leishmaniasis. The main mechanisms involved in the development and permanence of this pathology are linked to evasion of the immune response. Crosstalk between the immune system and particularities of each pathogenic species is associated with diverse disease manifestations. Lipophosphoglycan (LPG), one of the most important molecules present on the surface of Leishmania parasites, is divided into four regions with high molecular variability. Although LPG plays an important role in host-pathogen and vector-parasite interactions, the distribution and phylogenetic relatedness of the genes responsible for its synthesis remain poorly explored. The recent availability of full genomes and transcriptomes of Leishmania parasites offers an opportunity to leverage insight on how LPG-related genes are distributed and expressed by these pathogens. RESULTS: Using a phylogenomics-based framework, we identified a catalog of genes involved in LPG biosynthesis across 22 species of Leishmania from the subgenera Viannia and Leishmania, as well as 5 non-Leishmania trypanosomatids. The evolutionary relationships of these genes across species were also evaluated. Nine genes related to the production of the glycosylphosphatidylinositol (GPI)-anchor were highly conserved among compared species, whereas 22 genes related to the synthesis of the repeat unit presented variable conservation. Extensive gain/loss events were verified, particularly in genes SCG1-4 and SCA1-2. These genes act, respectively, on the synthesis of the side chain attached to phosphoglycans and in the transfer of arabinose residues. Phylogenetic analyses disclosed evolutionary patterns reflective of differences in host specialization, geographic origin and disease manifestation. CONCLUSIONS: The multiple gene gain/loss events identified by genomic data mining help to explain some of the observed intra- and interspecies variation in LPG structure. Collectively, our results provide a comprehensive catalog that details how LPG-related genes evolved in the Leishmania parasite specialization process.

Tamoxifen inhibits the biosynthesis of inositolphosphorylceramide in Leishmania.

Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [3H]-sphingosine and myo-[3H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn-1-O-(C18:0)alkyl -2-O-(C18:1)acylglycerol-3-HPO4-inositol and sn-1-O-(C18:0)acyl-2-O-(C18:1)acylglycerol-3-HPO4-inositol) species. Substrate saturation kinetics of myo-inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC50 value of 8.48 µM (95% CI 7.68-9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites.

Promoter identification and analysis of key glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Glycosphingolipid synthesis is essential for MDCK cell differentiation.

Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway.

Cellular and molecular biology of glycosphingolipid glycosylation.

Brain tissue is characterized by its high glycosphingolipid content, particularly those containing sialic acid (gangliosides). As a result of this observation, brain tissue was a focus for studies leading to the characterization of the enzymes participating in ganglioside biosynthesis, and their participation in driving the compositional changes that occur in glycolipid expression during brain development. Later on, this focus shifted to the study of cellular aspects of the synthesis, which lead to the identification of the site of synthesis in the neuronal soma and their axonal transport toward the periphery. In this review article, we will focus in subcellular aspects of the biosynthesis of glycosphingolipid oligosaccharides, particularly the mechanisms underlying the trafficking of glycosphingolipid glycosyltransferases from the endoplasmic reticulum to the Golgi, those that promote their retention in the Golgi and those that participate in their topological organization as part of the complex membrane bound machinery for the synthesis of glycosphingolipids.

Inositolphosphoceramide metabolism in Trypanosoma cruzi as compared with other trypanosomatids.

Chagas disease is caused by Trypanosoma cruzi and is endemic to North, Central and South American countries. Current therapy against this disease is only partially effective and produces adverse side effects. Studies on the metabolic pathways of T. cruzi, in particular those with no equivalent in mammalian cells, might identify targets for the development of new drugs. Ceramide is metabolized to inositolphosphoceramide (IPC) in T. cruzi and other kinetoplastid protists whereas in mammals it is mainly incorporated into sphingomyelin. In T. cruzi, in contrast to Trypanosoma brucei and Leishmania spp., IPC functions as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs). Inhibition of IPC and GIPLs biosynthesis impairs differentiation of trypomastigotes into the intracellular amastigote forms. The gene encoding IPC synthase in T. cruzi has been identified and the enzyme has been expressed in a cell-free system. The enzyme involved in IPC degradation and the remodelases responsible for the incorporation of ceramide into free GIPLs or into the glycosylphosphatidylinositols anchoring glycoproteins, and in fatty acid modifications of these molecules of T. cruzi have been understudied. Inositolphosphoceramide metabolism and remodeling could be exploited as targets for Chagas disease chemotherapy.

Neobiosynthesis of glycosphingolipids by plasma membrane-associated glycosyltransferases.

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.

UV-MALDI mass spectrometry analysis of NBD-glycosphingolipids without an external matrix.

Each day, advances in the instrumentation and operating protocols bring new applications and insights into the molecular processes of ultra violet-matrix assisted laser desorption/ionization-mass spectrometry (UV-MALDI MS), increasing its potential use. We report here an approach in which mass spectrometry analysis of sphingolipids has been performed using a fluorescent tag (nitrobenz-2-oxa-1, 3-diazole, NBD) covalently linked to the sphingoid base as matrix. Thus, different labeled-sphingolipids were analyzed: ceramide, dihydroceramide, acetylceramide, glucosylceramide, galactosylceramide, galactosyldihydroceramide. In addition an extract of glycosphingolipids obtained from epimastigote forms of Trypanosoma cruzi metabolically labeled with NBD-ceramide was analyzed. The goal of this work is to show that no matrix needs to be added for the mass spectrometry analysis as the same tag used to label the lipids may generate efficiently analyte ions to obtain high quality signals.

Cloning and functional characterization of two key enzymes of glycosphingolipid biosynthesis in the amphibian Xenopus laevis.

Gangliosides are a subfamily of complex glycosphingolipids (GSLs) with important roles in many biological processes. In this study, we report the cDNA cloning, functional characterization, and the spatial and temporal expression of Xlcgt and Xlgd3 synthase during Xenopus laevis development. Xlcgt was expressed both maternally and zigotically persisting at least until stage 35. Maternal Xlgd3 synthase mRNA could not be detected and showed a steady-state expression from gastrula to late tailbud stage. Xlcgt is mainly present in involuted paraxial mesoderm, neural folds, and their derivatives. Xlgd3 synthase transcripts were detected in the dorsal blastoporal lip, in the presumptive neuroectoderm, and later in the head region, branchial arches, otic and optic primordia. We determined the effect of glycosphingolipid depletion with 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP) in mesodermal layer. PPMP-injected embryos showed altered expression domains in the mesodermal markers. Our results suggest that GSL are involved in convergent-extension movements during early development in Xenopus.

Inhibition of Leishmania (Leishmania) amazonensis growth and infectivity by aureobasidin A.

OBJECTIVES: To study the effect of aureobasidin A, an inhibitor of inositol phosphorylceramide (IPC) synthase, on Leishmania growth and infectivity. METHODS: Effects of aureobasidin A were determined for: (i) promastigote growth in axenic culture; (ii) promastigote infectivity in macrophage monolayers; (iii) development of footpad lesions in BALB/c mice; (iv) differentiation of amastigotes into promastigotes. RESULTS: Aureobasidin A (20 microM) inhibited 90% of Leishmania (Leishmania) amazonensis promastigote growth in axenic culture, but the parasites remained viable, i.e. growth curves returned to normal after aureobasidin A was removed from culture medium. The aureobasidin A IC50 was determined by MTT assay as 4.1 microM for L. (L.) amazonensis promastigotes, 12.6 microM for Leishmania (Leishmania) major and 13.7 microM for Leishmania (Viannia) braziliensis. There was a significant delay in infection when L. (L.) amazonensis promastigotes pre-treated with aureobasidin A were inoculated into BALB/c mouse footpads. When aureobasidin A was added to cultured macrophages infected with amastigotes, the number of infected macrophages was reduced by >90%. CONCLUSIONS: Aureobasidin A is an interesting pharmacological tool to investigate the effect of lipid metabolism inhibition in Leishmania spp.

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis.

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.

A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana.

Recognition of pathogen-associated molecular patterns (PAMP) influences the response of dendritic cells (DC) and therefore development of innate and adaptive immunity. Different forms of Leishmania mexicana have distinct effects on DC, with promastigotes and amastigotes being activating and apparently neutral, respectively. We investigated whether stage-specific differences in surface composition might account for these distinct effects. Amastigotes and promastigotes lacking the lpg1 gene needed for lipophosphoglycan (LPG) biosynthesis could not activate DC in vitro. Genome-wide transcriptional profiling of DC infected with wild-type or mutant promastigotes or wild-type amastigotes revealed that wild-type promastigotes induce an inflammatory signature that is lacking in DC exposed to the other parasite forms. The proinflammatory response pattern was partly recovered by reconstitution of lpg1 expression in lpg1-/- parasites, and exposure to purified LPG increased the expression of MHC class II and CD86 on DC. Infection with wild-type but not lpg1-/- promastigotes increased the number of activated DC in draining lymph nodes, and this was correlated with lower early parasite burdens in wild-type-infected animals. These in vivo and in vitro results suggest an LPG-dependent activation of DC that contributes to host defense and agree with the notion that the parasites evolved under immune pressure to down-regulate PAMP expression in mammalian hosts.

Inhibition of the synthesis of glycosphingolipid by a ceramide analogue (PPMP) in the gastrulation of Bufo arenarum.

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 microM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.

Carbohydrate and LPG expression in Leishmania viannia subgenus.

Glycosylated molecules expressed on the cell surface of Leishmania promastigotes contribute to the outcome of contact between the parasite and its invertebrate and vertebrate hosts. The expression of several such molecules is growth phase dependent. Information on the expression of carbohydrates by Leishmania of the Viannia subgenus (braziliensis complex), a widespread cause of morbidity in the Americas, is fragmentary. We have examined the relationship between growth phase and the expression of glycosylated surface structures in WHO reference strains of 3 species of the Viannia subgenus, i.e., L. panamensis, L. guyanensis, and L. braziliensis. Agglutination with lectins and the monoclonal antibody specific for the repeat unit of L. donovani lipophosphoglycan, CA7AE, distinguished logarithmic and stationary-phase promastigotes of all 3 species. Flow cytometry revealed increased heterogeneity and disparity in the expression of the repeat unit epitope in stationary-as compared to logarithmic-phase promastigotes. Biochemical analyses showed the LPG repeat unit of all 3 species reference strains to be constituted by mannose and galactose with little or no substitution and, hence, to be similar to the LPG of L. donovani. Initial quantitative analyses of L. braziliensis LPG indicated a 10-fold lower quantity of LPG in this species than L. donovani and an increase in the size of LPG in the stationary phase. These findings provide bases for isolating and biologically characterizing phenotypically distinct populations of promastigotes and for identifying molecular determinants of the host parasite-relationship among Leishmania Viannia.

Ontogenic development of membrane lipids in the chick optic lobe.

The developmental profiles of the lipid composition and their de novo synthesis and remodelling in the optic lobe of the chicken were studied. The 32P incorporation to phospholipids showed an active de novo synthesis mainly of phosphatidylinositol and of a particular fraction of phosphatidylcholine during the early stages of the embryo development, concomitantly with the beginning of synaptogenesis. This de novo synthesis of phospholipids strongly increased at hatching. On the other hand, phosphatidylinositol presented an active lipid exchange (acylation-deacylation) in the early stages of embryogenesis, indicating a strong incorporation of 14C-arachidonic acid during this period, followed by a fast drop in specific activity. Two different fractions of phosphatidylcholine were isolated by high-performance thin-layer chromatography with a different profile of fatty acid composition, disclosing their different physicochemical behavior, metabolic activities and evolution during embryogenesis. 32P incorporation into phosphatidylethanolamine remained very low during the earliest stages of embryogenesis, showing an increase when the process of synaptogenesis began, until hatching, when radioactivity reached a plateau. 14C-arachidonic acid incorporation into phosphatidylethanolamine was minimal. Furthermore, the phosphatidylethanolamine pool was progressively enriched in its ethanolamine plasmalogen throughout the development. Chromatographic analysis of lipid extracts showed the presence of cerebroside traces after 16 days of embryo incubation. At hatching, a remarkable increase in non-hydroxylated cerebrosides was observed concurrently with the appearance of hydroxylated ones. These glycosphingolipids, as well as the sulfatides, were markedly increased in the lipid extracts of optic lobes of adult animals, indicating the progressive development and maturity of the myelin sheath.

Inhibition of glycosylphosphatidylinositol biosynthesis in Leishmania mexicana by mannosamine.

In several Leishmania species, glycosylated inositol phospholipids exist as free lipids, as membrane protein anchors, and as the membrane-binding moieties of the lipophosphoglycans. Both the glycolipid-anchored cell surface metalloproteinase, gp63, and the lipophosphoglycans have been proposed to be involved in cell invasion. Moreover, the lipophosphoglycans have been implicated in the survival of Leishmania in the parasitophorous vacuole of the host macrophage. In this report we show that mannosamine effectively inhibits the biosynthesis of both free glycosylated inositol phospholipids and the lipophosphoglycans of Leishmania mexicana. [3H]Mannosamine is incorporated into glycosylated inositol phospholipids, but not significantly into lipophosphoglycans when added as a radiochemical tracer at a subinhibitory concentration. The reversible inhibitory effect of mannosamine may be useful for studying precursor/product relationships during the biosynthesis of free glycosylated inositol phospholipids, glycolipid anchors, and the lipophosphoglycans. The implications of these data for the mode of action of mannosamine are discussed.

Expression of lipophosphoglycan, high-molecular weight phosphoglycan and glycoprotein 63 in promastigotes and amastigotes of Leishmania mexicana.

The abundant surface glycoconjugate of Leishmania promastigotes, lipophosphoglycan (LPG), forms a blue-colored complex (lambda max = 649 nm) with the cationic dye Stains-all, which can be quantitated densitometrically on polyacrylamide gels of cell lysates. Promastigotes of Leishmania mexicana, Leishmania major and Leishmania donovani yield values of 1-3 x 10(6) LPG molecules cell-1. In amastigotes the LPG content is down-regulated below the detection limit (< 10(3) molecules cell-1) in L. mexicana and L. donovani, but remains significant in L. major (2 x 10(3) molecules cell-1). In the case of L. mexicana, these results are supported by immunological studies. Using several monoclonal and polyclonal antibodies, LPG is undetectable by immunoblotting in lysates of either amastigotes or infected macrophages and the amastigote surface is devoid of LPG as judged by immunofluorescence and immunoelectron microscopy. Immunoblotting experiments demonstrate that amastigotes synthesize hydrophilic high-molecular weight compounds which stain blue with Stains-all and cross-react with the monoclonal and polyvalent antibodies suggesting the presence of similar phosphoglycan structures as in LPG. The high-molecular weight phosphoglycan appears to be located in the lumen of the flagellar pocket of mouse lesion amastigotes and may be secreted from there into the lumen of the parasitophorous vacuole of parasitized macrophages. In L. mexicana promastigotes the surface protease gp63 is amphiphilic and comprises about 1% of the cellular proteins. In contrast, in amastigotes gp63-related proteins are predominantly hydrophilic; they amount to only about 0.1% of the cellular proteins and are mainly located in the lumen of the extended lysosomes (megasomes) characteristic for this species.