Stage-specific binding of Leishmania donovani to the sand fly vector midgut is regulated by conformational changes in the abundant surface lipophosphoglycan.

The life cycle of Leishmania parasites within the sand fly vector includes the development of extracellular promastigotes from a noninfective, procyclic stage into an infective, metacyclic stage that is uniquely adapted for transmission by the fly and survival in the vertebrate host. These adaptations were explored in the context of the structure and function of the abundant surface lipophosphoglycan (LPG) on Leishmania donovani promastigotes. During metacyclogenesis, the salient structural feature of L. donovani LPG is conserved, involving expression of a phosphoglycan chain made up of unsubstituted disaccharide-phosphate repeats. Two important developmental modifications were also observed. First, the size of the molecule is substantially increased because of a twofold increase in the number of phosphorylated disaccharide repeat units expressed. Second, there is a concomitant decrease in the presentation of terminally exposed sugars. This later property was indicated by the reduced accessibility of terminal galactose residues to galactose oxidase and the loss of binding by the lectins, peanut agglutinin, and concanavalin A, to metacyclic LPG in vivo and in vitro. The loss of lectin binding was not due to downregulation of the capping oligosaccharides as the same beta-linked galactose or alpha-linked mannose-terminating oligosaccharides were present in both procyclic and metacyclic promastigotes. The capping sugars on procyclic LPG were found to mediate procyclic attachment to the sand fly midgut, whereas these same sugars on metacyclic LPG failed to mediate metacyclic binding. And whereas intact metacyclic LPG did not inhibit procyclic attachment, depolymerized LPG inhibited as well as procyclic LPG, demonstrating that the ligands are normally buried. The masking of the terminal sugars is attributed to folding and clustering of the extended phosphoglycan chains, which form densely distributed particulate structures visible on fracture-flip preparations of the metacyclic surface. The exposure and subsequent masking of the terminal capping sugars explains the stage specificity of promastigote attachment to and release from the vector midgut, which are key events in the development of transmissible infections in the fly.

Stage-specific adhesion of Leishmania promastigotes to the sandfly midgut.

Although leishmaniasis is transmitted to humans almost exclusively by the bite of infected phlebotomine sandflies, little is known about the molecules controlling the survival and development of Leishmania parasites in their insect vectors. Adhesion of Leishmania promastigotes to the midgut epithelial cells of the sandfly was found to be an inherent property of noninfective-stage promastigotes, which was lost during their transformation to metacyclic forms, thus permitting the selective release of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release was found to be controlled by specific developmental modifications in terminally exposed saccharides on lipophosphoglycan, the major surface molecule on Leishmania promastigotes.

CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells.

Immunoglobulin G (IgG) responses require major histocompatibility complex (MHC)-restricted recognition of peptide fragments by conventional CD4(+) helper T cells. Immunoglobulin G responses to glycosylphosphatidylinositol (GPI)- anchored protein antigens, however, were found to be regulated in part through CD1d-restricted recognition of the GPI moiety by thymus-dependent, interleukin-4-producing CD4(+), natural killer cell antigen 1.1 [(NK1.1)+] helper T cells. The CD1-NKT cell pathway regulated immunogobulin G responses to the GPI-anchored surface antigens of Plasmodium and Trypanosoma and may be a general mechanism for rapid, MHC-unrestricted antibody responses to diverse pathogens.

Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins.

The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana.

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.

Comparative Metabolomics of Mycoplasma bovis and Mycoplasma gallisepticum Reveals Fundamental Differences in Active Metabolic Pathways and Suggests Novel Gene Annotations.

Mycoplasmas are simple, but successful parasites that have the smallest genome of any free-living cell and are thought to have a highly streamlined cellular metabolism. Here, we have undertaken a detailed metabolomic analysis of two species, Mycoplasma bovis and Mycoplasma gallisepticum, which cause economically important diseases in cattle and poultry, respectively. Untargeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses of mycoplasma metabolite extracts revealed significant differences in the steady-state levels of many metabolites in central carbon metabolism, while 13C stable isotope labeling studies revealed marked differences in carbon source utilization. These data were mapped onto in silico metabolic networks predicted from genome wide annotations. The analyses elucidated distinct differences, including a clear difference in glucose utilization, with a marked decrease in glucose uptake and glycolysis in M. bovis compared to M. gallisepticum, which may reflect differing host nutrient availabilities. The 13C-labeling patterns also revealed several functional metabolic pathways that were previously unannotated in these species, allowing us to assign putative enzyme functions to the products of a number of genes of unknown function, especially in M. bovis. This study demonstrates the considerable potential of metabolomic analyses to assist in characterizing significant differences in the metabolism of different bacterial species and in improving genome annotation. IMPORTANCE Mycoplasmas are pathogenic bacteria that cause serious chronic infections in production animals, resulting in considerable losses worldwide, as well as causing disease in humans. These bacteria have extremely reduced genomes and are thought to have limited metabolic flexibility, even though they are highly successful persistent parasites in a diverse number of species. The extent to which different Mycoplasma species are capable of catabolizing host carbon sources and nutrients, or synthesizing essential metabolites, remains poorly defined. We have used advanced metabolomic techniques to identify metabolic pathways that are active in two species of Mycoplasma that infect distinct hosts (poultry and cattle). We show that these species exhibit marked differences in metabolite steady-state levels and carbon source utilization. This information has been used to functionally characterize previously unknown genes in the genomes of these pathogens. These species-specific differences are likely to reflect important differences in host nutrient levels and pathogenic mechanisms.

Using metabolomics to dissect host-parasite interactions.

Protozoan parasites have evolved diverse growth and metabolic strategies for surviving and proliferating within different extracellular and intracellular niches in their mammalian hosts. Metabolomic approaches, including high coverage metabolite profiling and (13)C/(2)H-stable isotope labeling, are increasingly being used to identify parasite metabolic pathways that are important for survival and replication in vivo. These approaches are highlighting new links between parasite carbon metabolism and the ability of different parasite stages to colonize specific niches or host cell types. They have also revealed novel metabolic regulatory mechanisms that are important for homeostasis and survival in potentially nutrient variable environments. These studies highlight the importance of parasite and host metabolism as determinants of host-parasite interactions.

The major surface antigens of Entamoeba histolytica trophozoites are GPI-anchored proteophosphoglycans.

Trophozoites of the parasitic protozoa, Entamoeba histolytica, synthesize a cell surface lipoglycoconjugate, termed lipophosphoglycan, which is thought to be an important virulence factor and potential vaccine candidate against invasive amebiasis. Here, we show that the E. histolytica lipophosphoglycans are in fact glycosylphosphatidylinositol (GPI)-anchored proteophosphoglycans (PPGs). These PPGs contain a highly acidic polypeptide component which is rich in Asp, Glu and phosphoserine residues. This polypeptide component is extensively modified with linear glycan chains having the general structure, [Glcalpha1-6](n)Glcbeta1-6Gal (where n=2-23). These glycan chains can be released after mild-acid hydrolysis with trifluoroacetic or hydrofluoric acid and are probably attached to phosphoserine residues in the polypeptide backbone. The PPGs are further modified with a GPI anchor which differs from all other eukaryotic GPI anchors so far characterized in containing a glycan core with the structure, Gal(1)Man(2)GlcN-myo-inositol, and in being heterogeneously modified with chains of alpha-galactose. Trophozoites of the pathogenic HM-1:IMSS strain synthesize two distinct classes of PPG which have polydisperse molecular masses of 50-180 kDa (PPG-1) and 35-60 kDa (PPG-2) and are modified with glucan side-chains of different average lengths. In contrast, the non-pathogenic Rahman strain synthesizes one class of PPG which is only elaborated with short disaccharide side-chains (i.e. Glcbeta1-6Gal). However, the PPGs are abundant in all strains (8x10(7) copies per cell) and are likely to form a protective surface coat.

The glycoinositolphospholipids from Leishmania panamensis contain unusual glycan and lipid moieties.

The cell surface of Leishmania parasites is coated by glycosylphosphatidylinositol (GPI)-anchored macromolecules (glycoproteins and a lipophosphoglycan) and a polymorphic family of free GPI glycolipids or glycoinositolphospholipids (GIPLs). Here we show that GIPLs with unusual glycan and lipid moieties are likely to be major cell surface components of L. panamensis (subgenus Viannia) promastigotes. These glycolipids were purified by high performance thin layer chromatography and their structures determined by gas-liquid chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, methylation analysis and chemical and enzymatic sequencing of the glycan headgroups. The major GIPLs contained two glycan core sequences, Manalpha1-3Manalpha1-4GlcN-phosphatidylinositol (type-2 series) or Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1- 4GlcN-phosphatidylinosit ol (hybrid series), which were elaborated with Galalpha1-2Galbeta1- or Galalpha1-2/3Galalpha1-2Galbeta1- extensions that were attached to the 3-position of the alpha1-3 linked mannose. The phosphatidylinositol moiety contained exclusively diacylglycerol with palmitoyl, stearoyl and heptadecanoyl chains. Non-galactosylated GIPL species with the same core structures were also found. The galactose extensions and the presence of diacylglycerol in the lipid moieties are novel features for the GIPLs of Leishmania spp. The implications of these structures for the biosynthesis of leishmanial GIPLs and their putative function in the mammalian host are discussed.

Purification, partial characterization and immunolocalization of a proteophosphoglycan secreted by Leishmania mexicana amastigotes.

The intracellular amastigote form of the parasitic protozoon Leishmania mexicana expresses a high-molecular weight phosphoglycan, which is antigenically related to the surface glycolipid lipophosphoglycan and the secreted enzyme acid phosphatase of Leishmania promastigotes. This antigen was purified from a cell-free homogenate of infected mouse tissue and from amastigotes. Compositional and immunological analysis of the purified components indicate a proteophosphoglycan structure consisting of serine-rich polypeptide chains and mild acid-labile phosphooligosaccharides capped by mannooligosaccharides. Immunofluorescence and immunoelectron microscopy of parasitized mouse peritoneal macrophages and infected mouse tissue suggest that the proteophosphoglycan is secreted in large amounts by amastigotes via their flagellar pockets into the parasitophorous vacuoles of host cells. In some infected macrophages proteophosphoglycan is also located in vesicles apparently originating from the parasitophorous vacuole, which demonstrates redistribution of a secreted amastigote antigen in parasitized host cells.

The glycoinositolphospholipid profiles of two Leishmania major strains that differ in lipophosphoglycan expression.

The glycolipid profiles of two Leishmania major strains which differ in their expression of the major glycoconjugate, lipophosphoglycan (LPG), have been compared. All the glycolipids in these strains belong to a class of glycoinositolphospholipids (GIPLs) which can be metabolically labelled with [3H]inositol and are sensitive to phosphatidylinositol-specific phospholipase C. The major glycolipids in the LPG-producing L. major strain V121 are tetraglycosyl phosphatidylinositol (GIPL-1), pentaglycosyl phosphatidylinositol (GIPL-2), hexaglycosyl phosphatidylinositol (GIPL-3) and lyso-GIPL-3. These were identified by their sensitivity to lipases, and by BioGel P4 chromatography of the glycan fragments released after nitrous acid deamination. Similar glycolipids are also present in the LPG-deficient L. major strain LRC-L119. However, this strain also produces several highly polar GIPLs (GIPL-4, 5 and 6) which are absent from V121 and which may represent truncated forms of LPG. Evidence is presented showing that the LPG and GIPLs from both strains contain galactofuranose, based on identification of labelled arabinose after mild periodate oxidation and reduction with NaB [3H]4. Furthermore, analysis of the deaminated glycan moieties after mild acid hydrolysis suggests that the GIPLs and the glycolipid anchor of LPG contain a common glycan core, which includes the galactofuranose. Finally, radiolabelling of intact cells indicates that there is restricted expression of some of the GIPLs in the plasma membrane and that the GIPL-2 is the predominant cell surface glycolipid. These data are consistent with some, but not all, of the GIPLs in V121 having a major role as precursors to the glycolipid core of LPG.

Recognition of the major cell surface glycoconjugates of Leishmania parasites by the human serum mannan-binding protein.

Activation of complement on the surface of parasitic protozoa of the genus Leishmania appears to be important for parasite infectivity in the mammalian host, as it allows these parasites to attach to and invade macrophages via their surface complement receptors. Serum mannan-binding protein (MBP) is a known activator of complement. Therefore, in the present study, we have investigated whether serum MBP binds to live Leishmania parasites, and to mannose-containing saccharides derived from the parasite cell surface. We have observed by fluorescence microscopy that biotinylated MBP binds to the surface of L. major and L. mexicana promastigotes. At this developmental stage the parasites are coated by a mannose-containing lipophosphoglycan (LPG). We have observed that radioiodinated MBP binds in a mannose-inhibitable manner to purified LPG which has been immobilized in plastic microwells, as well as to purified mannose-terminating di-, tri- and tetrasaccharide fragments ('cap' structures) which have been released by mild acid hydrolysis from the outer chains of the LPG, converted into neoglycolipids and resolved by thin-layer chromatography. 125I-MBP also binds in the chromatogram-binding assay to the mannose-containing glycoinositol-phospholipids that are expressed in high copy number on both the promastigote and the intracellular amastigote stages of most Leishmania species. These data suggest that MBP has the potential to opsonize the major developmental stages of Leishmania parasites, and provide a possible mechanism for the antibody-independent activation of complement on their surface.

Lipophosphoglycan expression and virulence in ricin-resistant variants of Leishmania major.

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats jointed by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the Mr 63,000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies. All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.

Function and assembly of the Leishmania surface coat.

Like many trypanosomatids, the cell surface coat of Leishmania spp. is responsible for mediating various host-parasite interactions as well as acting as a dense physical barrier. This confers protection to the parasites in the hostile environments of the sandfly midgut and the macrophage phagolysosome. The major components of the surface coat are tethered to the cell surface via glycosylphosphatidylinositol glycolipids, and the composition of this surface coat is exquisitely regulated during the course of the parasite life-cycle. In this paper, we review what is known about the composition, biosynthesis and function of these glycosylphosphatidylinositol-containing molecules found within the parasite surface coat.

The developmental regulation and biosynthesis of GPI-related structures in Leishmania parasites.

Most macromolecules on the surface of Leishmania parasites, including the major surface proteins and a complex lipophosphoglycan (LPG) are anchored to the plasma membrane via GPI glycolipids. Free glycoinositol-phospholipids (GIPLs) which are not linked to protein or phosphoglycan are also abundant in the plasma membrane. From structural and metabolic labeling studies it is proposed that most Leishmania species express three distinct pathways of GPI biosynthesis. Some of these pathways (i.e. those involved in the protein and LPG anchor biosynthesis) are down-regulated during the differentiation of the insect (promastigote) stage to the mammalian (amastigote) stage. In contrast, the GIPLs are expressed in high copy number in both developmental stages. Based on analysis of the lipid moieties of the different GPI species it is possible that the pathways of GPI anchor and GIPL biosynthesis are located in different subcellular compartments. The relative flux through the GIPL and LPG biosynthetic pathways has been examined in L. major promastigotes. These studies showed that while the rate of synthesis of the GIPLs and LPG is similar, LPG is shed more rapidly from the plasma membrane and has a higher turnover. The possible metabolic relationship between the GIPL and LPG biosynthetic pathways is discussed.

The surface glycoconjugates of parasitic protozoa: potential targets for new drugs.

Protozoan parasites are the cause of many disease in humans and their domestic livestock. Glycoconjugates (i.e. glycoproteins, glycolipids) on the cell surface of these extremely diverse and very primative eukaryotes play a crucial role in determining the specificity of the host-parasite interaction and in protecting the parasites within their respective hosts. These molecules frequently share a common structural feature in that they are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) glycolipid. While GPI protein-membrane anchors are ubiquitous among the eukaryotes, they are used with exceptionally high frequency in the protozoa. Some kinetopastid parasites also synthesise very high levels of GPI-related glycolipids that are not linked to protein. Thus GPI-anchored molecules or free GPI glycolipids send to dominate the cell surface molecular architecture of these organisms. The highly elevated levels and specialised nature of GPI metabolism in the kinetoplastid and other parasites suggests that the GPI biosynthetic pathway might be a good target for the development of new chemotherapeutic agents. This article reviews the wide range of functions that GPI protein anchors and GPI-related glycolipids are thought to perform in these organisms and some aspects of their biosynthesis.

A family of glycoinositol phospholipids from Leishmania major. Isolation, characterization, and antigenicity.

The glycolipids of the protozoan Leishmania major strain LRC-L119 belong to a class of glycoinositol phospholipids (GIPL) that show partial structural homology to the phosphatidylinositol-containing glycolipid membrane anchors of several eukaryotic proteins and the lipid moiety of L. major lipophosphoglycan. The GIPLs were the only glycolipids detected and were purified by octyl-Sepharose and thin layer chromatographies. Analysis of the native and dephosphorylated glycolipids (GIPLs 1-6) by gas chromatography-mass spectrometry revealed that the glycan moieties have between 4 and 10 saccharide residues and all contain mannose, galactose, and non-N-acetylated glucosamine. Some of the GIPLs also contain glucose (GIPL-6) and hexose monophosphate residues (GIPL 4-6). The presence of an inositol phospholipid moiety in all the GIPLs is indicated by the identification of 1 myo-inositol monophosphate residue/molecule and their susceptibility to phosphatidylinositol-specific phospholipase C. However, heterogeneity in the lipid moieties is indicated by differences in the compositional analysis and the behavior of the GIPLs on the thin layer chromatography after mild alkali hydrolysis or phospholipase A2 treatment. These results demonstrate that GIPLs 1-4 contain 1-alkyl-2-acylglycerol composed of saturated unbranched alkyl chains with carbon chain lengths of 18-26 and acyl chains of myristate, palmitate and stearate, whereas GIPL-5 and -6 contain lyso-alkylglycerol composed of mainly C24:0 and C26:0 alkyl chains. Analysis of the products of nitrous acid deamination demonstrates that these glycerolipids are present as alkylacylphosphatidylinositol (GIPLs 1-4) and 1-O-alkylglycerophosphoinositol (GIPL-5 and -6), respectively. GIPL-2 and -3 are labeled on the surface of living promastigotes with galactose oxidase/NaB[3H]4. These GIPLs also react with three monoclonal antibodies that recognize the surface of promastigotes and amastigotes of L. major and other Leishmania spp.

Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (review).

Glycosylphosphatidylinositols (GPIs) represent an abundant and ubiquitous class of eukaryotic glycolipids. Although these structures were originally discovered in the form of GPI-anchored cell surface glycoproteins, it is becoming increasingly clear that a significant proportion of the GPI synthetic output of a cell is not directed to protein anchoring. Indeed, pools of non-protein-linked GPIs can approach 10(7) molecules per cell in some cell types, especially the protozoa, with a large proportion of these molecules being displayed at the cell surface. Recent studies which form the subject of this review indicate that there is (a) considerable diversity in the range of structural modifications found on GPI glycolipids within and between species and cell types, (b) complexity in the topological arrangement of the GPI biosynthetic pathway in the endoplasmic reticulum, and (c) spatial restriction of the biosynthetic pathway within the endoplasmic reticulum. Furthermore, consistent with additional functional roles for these lipids beyond serving as protein anchor precursors, products of the GPI biosynthetic pathway appear to be widely distributed in the cellular endomembrane system. These studies indicate that there is still much to learn about the organization of glycolipid biosynthetic pathways in eukaryotic cells, the nature and subcellular distribution of the lipid products of these pathways, and the function of these lipids within cells.

Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis.