Glycoinositolphospholipids Research Articles

The α-Galactosyl Carbohydrate Epitope in Pathogenic Protozoa.

The α-gal epitope, which refers to the carbohydrate α-d-Galp-(1 → 3)-β-d-Galp-(1 → 4)-d-GlcNAc-R, was first described in the glycoconjugates of mammals other than humans. Evolution caused a mutation that resulted in the inactivation of the α-1,3-galactosyltransferase gene. For that reason, humans produce antibodies against α-d-Galp containing glycoproteins and glycolipids of other species. We summarize here the glycoconjugates with α-d-Galp structures in Trypanosoma, Leishmania, and Plasmodium pathogenic protozoa. These were identified in infective stages of Trypanosoma cruzi and in Plasmodium sporozoites. In Leishmania, α-d-Galp is linked differently in the glycans of glycoinositolphospholipids (GIPLs). Chemically synthesized neoglycoconjugates have been proposed as diagnostic tools and as antigens for vaccines. Several syntheses reported for the α-gal trisaccharide, also called the Galili epitope, and the glycans of GIPLs found in Leishmania, the preparation of neoglycoconjugates, and the studies in which they were involved are also included in this Review.

Specific Recognition of β-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients.

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients’ blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method’s limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal β-galactofuranosyl (β-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients’ sera react specifically with synthetic β-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfβ1,3Manpα-(CH2)3SH (glycan G29SH) and Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

Expanding the knowledge of the chemical structure of glycoconjugates from Trypanosoma cruzi TcI genotype. Contribution to taxonomic studies.

One of the main obstacles to the treatment of Chagas disease is the genetic and phenotypical variance displayed by T. cruzi strains, resulting in differences in morphology, virulence, pathogenicity and drug susceptibility. To better understand the role of glycoconjungates in Chagas disease, we performed the molecular characterization of the O-linked chains from mucins and glycoinositolphospholipids (GIPLs) of the Silvio X10 clone 1 strain. We demonstrated the presence of a β-galactofuranose (β-Galf) unity linked to the O-4 position of the α-N-acetylglucosamine (α-GlcNAc)O-4 in Tc-mucins. GIPLs analysis showed that the lipidic portion is exclusively composed of ceramide and the PI-oligossacharidic portion contains the Man4(AEP)GlcN-Ins-PO4 core, substituted by ethanolamine-phosphate (EtNP) on the third distal mannose from inositol, which may or may not have a terminal β Galf unity. These results confirm the classification of the Silvio X10/1 strain in group T. cruzi I. Again, it is noted that the study of T. cruzi surface glycoconjugates confirm the molecular results and the hypothesis that surface glycoconjugates may be interesting biomarker for the differentiation of trypanosomatid strains.

Analysis of the carbohydrate and lipid components of glycosylphosphatidylinositol structures.

AbstractGlycosylphosphatidylinositols (GPIs) are a family of structures that contain the structural motif: Manα1-4GlcNH2α1-6myo-Inositol-1-PO4-lipid. This common substructure suggests that this family of molecules are biosynthetically related and differentiates them from other glycosylated phosphoinositides, such as the glycosylated phosphatidylinositols of mycobacteria and the glycosylated inositol phosphoceramides of yeasts and plants. The GPI family can be conveniently divided into two groups based on structural homology and function. The first group (1–28) are the membrane protein anchors (Fig. 1) that are found covalently linked to the C-termini of a wide variety of externally disposed plasma membrane proteins throughout the eukaryotes These GPI anchors afford a stable attachment of proteins to the membrane and can be viewed as an alternative mechanism of membrane attachment to a single-pass hydrophobic transmembrane peptide domain. For recent reviews of GPI anchor structure, biosynthesis, and function see refs. 29–31. The second group of GPI structures have only been found in protozoan organisms. These molecules exist as free glycophospholipids, such as the glycoinositol phospholipids (GIPLs) of the Leishmania, Trypanosoma cruzi, Leptomonas, Herpetomonas, and Phytomonas (29,32–34), or attached to phosphorylated repeating units as in the lipophosphoglycans (LPGs) of the Leishmania (29,35). In this chapter protocols specifically designed to analyse the protein-linked GPI anchors, although they are also applicable to the GIPLs and, to some extent, the LPGs will be described. GPI anchor structures All GPI anchors attached to protein contain the conserved structure shown above with various substituents (R1–R5) and lipids, as indicated. Some structures contain an additional fatty acyl chain attached to the 2-position of the myoinositol ring. All metazoan organisms contain at least one, and sometimes two, extra ethanolamine phosphate (EtNPO4) substituents in addition to the one used as a bridge to the protein C-terminal amino acid. When a substituent is known to be attached to a certain sugar residue but the linkage position is unknown, this is indicated by a question mark. Square brackets are used to show substituents for which the site of attachment has not been determined. The ± symbol indicates that the associated residue is found on only a proportion of the structures AEP is 2-amino-ethyl-phosphonate. KeywordsSodium Acetate BufferHydrogen FluorideInositol RingExoglycosidase DigestionLabel GlycanThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Differential modulation of macrophage response elicited by glycoinositolphospholipids and lipophosphoglycan from Leishmania (Viannia) shawi

In this work, some aspects of the glycobiology of Leishmania shawi were examined, as it is a causative agent of cutaneous leishmaniasis in the New World. Additionally, the interaction of L. shawi's main glycoconjugates [lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs)] with macrophages was evaluated in vitro. L. shawi LPG was devoid of side-chains in its repeat units, whereas monosaccharide analysis showed that GIPLs were suggestive of mannose-rich (type I or hybrid). In order to evaluate the biological roles of those molecules, BALB/c resident peritoneal macrophages were incubated with these glycoconjugates for 24h, and the levels of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-12p70 and IL-10, were determined. In general, the GIPLs exhibited a greater proinflammatory role than the LPGs did. However, for the first time, the GIPLs from this species were able to trigger the production of IL-10, an anti-inflammatory cytokine. In conclusion, L. shawi glycoconjugates were able to interact with the innate immune compartment. These data reinforce the role of parasite glycoconjugates during parasite and host cell interactions.

Leishmania enriettii: biochemical characterisation of lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) and infectivity to Cavia porcellus.

BackgroundLeishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown.MethodsMice peritoneal macrophages from C57BL/6 and knock-out (TLR2 −/−, TLR4 −/−) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively.ResultsLPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO4. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF–α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE).ConclusionThe two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.

Ether‐Type Moieties in the Lipid Part of Glycoinositolphospholipids of Acanthamoeba rhysodes

Ether lipids were identified among components liberated with HF and nitrous acid deamination from Acanthamoeba rhysodes whole cells and its membrane glycoinositolphospholipids (GIPL). Liberated ether glycerols were converted to various derivatives that served characterization thereof. These included TMS and isopropylidene derivatives, oxidation with sodium periodate to aldehyde followed by reduction with NaBH4 to alcohol, and reaction of the alcohol with acetic anhydrite to form acetate derivatives. Periodate sensitivity demonstrated that the alkyl side chains were linked to the sn-1 position of glycerol. Combined information from TLC, GC–MS analysis, MALDI-TOF spectrometry, and chemical degradation experiments indicated the presence of ether-linked saturated normal and branched hydrocarbons with a length of C20–23 in the phospholipid fraction, C20–24 in free GPI, and C21–23 in the LPG polymer. The distribution of particular classes of alkylglycerols was similar for phospholipid and GPI fractions, and amounted to 2.62 % (±0.04–0.28) 1-O-eicosanyl-sn-glycerol, 16.66 % (±0.32–1.1) 1-O-uncosanyl-sn-glycerol, 9.18 % (±0.33–1.37) anteiso-1-O-docosanyl-sn-glycerol, 47.56 % (±0.32–2.14) 1-O-docosanyl-sn-glycerol, 20.56 % (±0.58–1.67) anteiso-1-O-tricosanyl-sn-glycerol, and 2.34 % (±0.12–0.63) 1-O-tricosanyl-sn-glycerol. For LPG preparation, the most abundant were anteiso-1-O-tricosanyl-sn-glycerol (57.26 %) and 1-O-docosanyl-sn-glycerol (30.12 %). The data from TLC and GC–MS analysis showed that ether lipids from phospholipids probably represent the lyso-alkylglycerol type, while those derived from GIPL are alkylacylglycerol moieties.

Leishmania mexicana Infection Induces IgG to Parasite Surface Glycoinositol Phospholipids that Can Induce IL-10 in Mice and Humans

Infection with the intracellular protozoan parasite Leishmania mexicana causes chronic disease in C57BL/6 mice, in which cutaneous lesions persist for many months with high parasite burdens (107–108 parasites). This chronic disease process requires host IL-10 and FcγRIII. When Leishmania amastigotes are released from cells, surface-bound IgG can induce IL-10 and suppress IL-12 production from macrophages. These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control. However, antibodies targets and the kinetics of antibody production are unknown. Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG. We now show that glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by mouse IgG1 by 6 weeks of infection, with a rapid increase between 12 and 16 weeks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII-deficient mice. A single prominent spot on TLC is recognized by IgG, and the glycolipid is a glycosyl phosphatidylinositol containing a branched mannose structure. We show that the lipid structure of the GIPL (the sn-2 fatty acid) is required for antibody recognition. This GIPL is abundant in L. mexicana amastigotes, rare in stationary-phase promastigotes, and absent in L. major, consistent with a role for antibodies to GIPLs in chronic disease. A mouse monoclonal anti-GIPL IgG recognizes GIPLs on the parasite surface, and induces IL-10 from macrophages. The current work also extends this mouse analysis to humans, finding that L. mexicana-infected humans with localized and diffuse cutaneous leishmaniasis have antibodies that recognize GIPLs, can bind to the surface of amastigotes, and can induce IL-10 from human monocytes. Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.

Trypanosoma cruzi Adjuvants Potentiate T Cell-Mediated Immunity Induced by a NY-ESO-1 Based Antitumor Vaccine

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4+ T and CD8+ T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-γ) production by CD4+ T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8+ T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4+ T and CD8+ T cell responses elicited by a specific immunological adjuvant.

Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: Modulation of Innate Immune System and Variations in Carbohydrate Structure

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2−/− and TLR4−/−) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages.

Leishmania mexicana infection induces antibody responses to parasite surface glycolipids that can induce IL-10 and suppress IL-12 from macrophages (37.25)

Abstract Chronic disease of mice caused by the protozoan parasite Leishmania mexicana requires IL-10 and FcγRIII. Immune complexes consisting of IgG bound to the surface of Leishmania amastigotes can induce IL-10 and suppress IL-12 production from macrophages. However, the targets of these antibodies and the kinetics of their production are not known. Several groups have attempted, without success, to identify surface proteins on the amastigote form of the parasite to which IgG can bind. Using ELISA and thin layer chromatography (TLC) immunoblot methods we now show that parasite glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by IgG1 by 6 weeks of infection with a rapid increase between 14-16 wks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII KO mice. We found that a single prominent spot on TLC is recognized by mouse serum of infected mice and that the glycolipid has a glycosyl phosphatidylinositol (GPI) structure as assessed by GPI-PLC and GPI-PLD susceptibility. Alpha-mannosidase treatment changed the mobility of the recognized glycolipid, indicating that a branched mannose occurs in the structure, but is not required for IgG binding. Further characterization of the target glycolipids will have important implications for vaccine development as well as elucidating the poorly understood role of glycolipids in immunology. Funded by VA Merit, the University of Pennsylvania, and NIH R01-AI081717.

Structures of Glycolipids Found in Trypanosomatids: Contribution to Parasite Functions

Neutral monohexosylceramides (CMHs) globosides (globotriasyl ceramides), other glycosphingolipids (GSLs) and more complex structures such as glycoinositol-phospholipids(GIPLs) and glycosyl phosphatidylinositol (GPI) anchors have been described in several members of the trypanosomatid family. These highly bioactive molecules are not only components of biological structures but also participants in host-parasite interactions such as macrophage invasion, antigenic presentation and signal transduction. Glycolipid structures have been studied using mass spectrometry (MS).This review describes a wide range of glycoconjugates with unique and complex structures that are present in several trypanosomatid species. Their structures are described in the context of their biological significance.

Trypanosoma cruzi: Involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus

Trypanosoma cruzi epimastigotes adhere in vivo to the luminal surface of their triatomid vector digestive tract by molecular mechanisms, as yet, unknown. Here, we show that the administration of 0.5 μM epimastigote major surface glycoinositolphospholipids (GIPLs) to the infected bloodmeal inhibits up to 90% parasite infection in Rhodnius prolixus. The parasite behavior was investigated in vitro using fragments of the insect midgut. The addition of GIPLs in concentration as low as 50–100 nM impaired 95% the attachment of epimastigotes. Previous treatment of GIPLs with trifluoroacetic acid to remove the terminal β-galactofuranosyl residues reversed 50% the epimastigote in vitro attachment. The binding sites of purified GIPLs on the luminal surface of the posterior midgut were exposed by immunofluorescence microscopy. These observations indicate that GIPLs are one of the components involved in the adhesion of T. cruzi to the luminal insect midgut surface and possibly one of the determinants of parasite infection in the insect vector.

Characterization of Leishmania (Viannia) braziliensis membrane microdomains, and their role in macrophage infectivity

Detergent-resistant membranes (DRMs) from Leishmania (Viannia) braziliensis promastigotes, insoluble in 1% Triton X-100 at 4 degrees C, were fractionated by sucrose density gradient ultracentrifugation. They were composed of glycoinositolphospholipids (GIPLs), inositol phosphorylceramide (IPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and sterols. In contrast, 1% Triton X-100-soluble fraction was composed of PE, phosphatidylcholine, phosphatidylserine, PI, IPC, sterol, and lyso-PI. High-performance thin-layer chromatography (HPTLC) immunostaining using monoclonal antibody SST-1 showed that 85% of GIPLs are present in DRMs, and immunoelectron microscopic analysis showed that SST-1-reactive components are located in patches along the parasite surface. No difference in GIPL pattern was observed by HPTLC between Triton X-100-soluble versus -insoluble fractions at 4 degrees C. Analysis of fatty acid composition in DRMs by GC-MS showed the presence of GIPLs containing an alkylacylglycerol, presenting mainly saturated acyl and alkyl chains. DRMs also contained sterol, IPC with saturated fatty acids, PI with at least one saturated acyl chain, and PE with predominantly oleic acid. Promastigotes treated with methyl-beta-cyclodextrin to disrupt lipid microdomains showed significantly lower macrophage infectivity, suggesting a relationship between lipid microdomains and the infectivity of these parasites.

Structural correlation of glycoinositol phospholipids and pathological spectra of leishmaniasis from the old world parasite isolates

Structural correlation of glycoinositol phospholipids and pathological spectra of leishmaniasis from the old world parasite isolates

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.

Structural Characterization of NETNES, a Novel Glycoconjugate in Trypanosoma cruzi Epimastigotes

The unicellular stercorarian protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. The epimastigote form of the parasite is covered in a dense coat of glycoinositol phospholipids and short glycosylphosphatidylinositol (GPI)-anchored mucinlike molecules. Here, we describe the purification and structural characterization of NETNES, a relatively minor but unusually complex glycoprotein that coexists with these major surface components. The mature glycoprotein is only 13 amino acids in length, with the sequence AQENETNESGSID, and exists in two forms with either four or five post-translational modifications. These are either one or two asparagine-linked oligomannose glycans, two linear alpha-mannose glycans linked to serine residues via phosphodiester linkages, and a GPI membrane anchor attached to the C-terminal aspartic acid residue. The variety and density of post-translational modifications on an unusually small peptide core make NETNES a unique type of glycoprotein. The N-glycans are predominantly Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-Asn; the phosphate-linked glycans are a mixture of (Manalpha1-2)0-3Man1-P-Ser; and the GPI anchor has the structure Manalpha1-2(ethanolamine phosphate)Manalpha1-2Manalpha1-6Manalpha1-4(2-aminoethylphosphonate-6)GlcNalpha1-6-myo-inositol-1-P-3(sn-1-O-(C16:0)alkyl-2-O-(C16:0)acylglycerol). Four putative NETNES genes were found in the T. cruzi genome data base. These genes are predicted to encode 65-amino acid proteins with cleavable 26-amino acid N-terminal signal peptides and 26-amino acid C-terminal GPI addition signal peptides.

Effect of glycoinositolphospholipids on the growth and differentiation ofTrypanosoma cruzisubpopulations

We have analyzed the biological effects of glycoinositolphospholipids (GIPLs) derived from Trypanosoma cruzi Chagas, 1909 isolates (G-645 and C-48) on the behavior of clone Dm 28c and the Y strain during their extracellular development in vitro and in the invertebrate host Rhodnius prolixus Stål, 1859. In vitro, a direct correlation between the presence of GIPLs and proliferation of both Dm 28c and the Y strain was observed. The GIPLs purified from strain G-645 induced higher differences in the levels of growth of clone Dm 28c when compared with GIPLs purified from C-48 or Leptomonas samueli Carvalho, 1973. Differentiation in the presence of G-645 GIPLs was significantly reduced (48 h), while a minor effect was observed with GIPLs from C-48 (72 h). Parasites adhered preferentially to areas on slides coated with GIPLs from G-645 but not GIPLs from C-48. Rhodnius prolixus fed with GIPLs from G-645 or paraformaldehyde-fixed G-645 parasites, coincident with or after establishment of Dm 28c infection, presented different growth and differentiation patterns compared with control bugs. Our data suggest the occurrence of intraspecific cooperation and (or) competition among the different subpopulations that make up a specific T. cruzi strain.

Expression of Functional TLR4 Confers Proinflammatory Responsiveness toTrypanosoma cruziGlycoinositolphospholipids and Higher Resistance to Infection withT. cruzi

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-kappaB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.

Glycoinositolphospholipid from Trypanosoma cruzi: Structure, Biosynthesis and Immunobiology

The pathogenic protozoan parasite Trypanosoma cruzi expresses on its surface an unusual family of glycoinositolphospholipids (GIPLs) closely related to glycosylphosphatidylinositol (GPI) anchors. Different parasite isolates express distinct GIPLs which fall into two series, depending on the substitution of the third mannosyl residue in the conserved glycan sequence Man4-(AEP)-GlcN-InsPO4 by ethanolamine phosphate or beta-galactofuranose. Although the exact role of these molecules in the cell biology and pathogenicity of T. cruzi remains unknown, the lipid and glycan moieties impart distinct responses to host T and B lymphocytes and phagocytes, overall favouring an immune response permissive to the parasite. The biosynsthesis of GIPLs follows a pathway similar to that observed for GPI anchors. However, a more detailed understanding might enable the development of specific inhibitors of parasite-specific enzymes and lead to novel drugs to ameliorate Chagas disease.