RESUMO
Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host-pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI-anchored proteins (GPI-APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well-characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N-terminal signal peptide and a single C-terminal transmembrane region containing a GPI anchor signal. Twenty-four GPI-anchored peptides were identified, of which nine are likely S. aucheniae-specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI-anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra- and inter-specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI-APs as drug targets and/or as vaccine or diagnostic antigens.
Assuntos
Camelídeos Americanos/parasitologia , Proteínas Ligadas por GPI/genética , Glicosilfosfatidilinositóis/metabolismo , Carne/parasitologia , Sarcocystis/imunologia , Sarcocistose/veterinária , Transcriptoma , Animais , Glicosilfosfatidilinositóis/química , Imunoterapia/veterinária , Filogenia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sarcocystis/genética , Sarcocystis/isolamento & purificação , Sarcocistose/parasitologia , Sarcocistose/terapia , Toxoplasma/enzimologia , Toxoplasma/genéticaRESUMO
BACKGROUND: Glycosylphosphatidylinositol (GPI) anchors are molecules located on cell membranes of all eukaryotic organisms. Proteins, enzymes, and other macromolecules which are anchored by GPIs are essential elements for interaction between cells, and are widely used by protozoan parasites when compared to higher eukaryotes. METHODS: More than one hundred references were collected to obtain broad information about mammalian and protozoan parasites' GPI structures, biosynthetic pathways, functions and attempts to use these molecules as drug targets against parasitic diseases. Differences between GPI among species were compared and highlighted. Strategies for drug discovery and development against protozoan GPI anchors were discussed based on what has been reported on literature. RESULTS: There are many evidences that GPI anchors are crucial for parasite's survival and interaction with hosts' cells. Despite all GPI anchors contain a conserved glycan core, they present variations regarding structural features and biosynthetic pathways between organisms, which could offer adequate selectivity to validate GPI anchors as drug targets. Discussion was developed with focus on the following parasites: Trypanosoma brucei, Trypanosoma cruzi, Leishmania, Plasmodium falciparum and Toxoplasma gondii, causative agents of tropical neglected diseases. CONCLUSION: This review debates the main variances between parasitic and mammalian GPI anchor biosynthesis and structures, as well as clues for strategic development for new anti-parasitic therapies based on GPI anchors.
Assuntos
Antiprotozoários/farmacologia , Glicosilfosfatidilinositóis/farmacologia , Leishmania/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Trypanosoma/efeitos dos fármacos , Animais , Antiprotozoários/química , Descoberta de Drogas , Glicosilfosfatidilinositóis/química , Humanos , Doenças Negligenciadas/tratamento farmacológicoRESUMO
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products-like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins)-are potent inducers of proinflammatory responses (i.e., cytokines and NO production) by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.
Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Glicoproteínas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Interleucina-12/metabolismo , Monócitos/metabolismo , Mucinas/química , Trypanosoma cruzi/imunologia , Células Cultivadas , Glicoproteínas/química , Glicosilfosfatidilinositóis/química , Humanos , Interferon gama/metabolismo , Ligação ProteicaRESUMO
Trypanosoma cruzi is a protozoan, responsible for Chagas disease, that parasites triatomines and some vertebrates, mainly Homo sapiens. In 2010, nearly 10 million people in whole world, most from Latin America, had Chagas disease, which is an illness of high morbidity, low mortality, and serious problems of quality of life. The available treatment has high toxicity and low efficacy at chronic phase. Some of the protozoan antigenic or virulence factors include complex carbohydrate structures that, due to their uniqueness, may constitute potential selective targets for the development of new treatments. One example of such structures is NETNES, a low abundance T. cruzi glycopeptide, comprising 13 amino acid residues, one or two N-glycosylation chains, a GPI anchor and two P-glycosylations. In this context, the current work aims to obtain an atomic model for NETNES, including its glycan chains and membrane attachment, in order to contribute in the characterization of its structure and dynamics. Based on POPC and GPI models built in agreement with experimental data, our results indicate that, in the first third of the simulation, NETNES peptide is very flexible in solution, bending itself between asparagine residues and lying down on some carbohydrates and membrane, exposing amino acid residues and some other glycans, mainly terminal mannoses, to the extracellular medium, remaining in this position until the end of simulations.
Assuntos
Glicoproteínas/química , Proteínas de Protozoários/química , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Proteínas de Protozoários/metabolismoRESUMO
The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Mucinas/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Citocinas/metabolismo , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/isolamento & purificação , Macrófagos Peritoneais/metabolismo , Mucinas/química , Mucinas/isolamento & purificação , Nitritos/metabolismo , Especificidade da EspécieRESUMO
A recent addition to the arsenal of tools for glycome analysis is the use of metabolic labels that allow covalent tagging of glycans with imaging probes. In this work we show that N-azidoglucosamine was successfully incorporated into glycolipidic structures of Plasmodium falciparum intraerythrocytic stages. The ability to tag glycoconjugates selectively with a fluorescent reporter group permits TLC detection of the glycolipids providing a new method to quantify dynamic changes in the glycosylation pattern and facilitating direct mass spectrometry analyses. Presence of glycosylphosphatidylinositol and glycosphingolipid structures was determined in the different extracts. Furthermore, the fluorescent tag was used as internal matrix for the MALDI experiment making even easier the analysis.
Assuntos
Eritrócitos/parasitologia , Glicolipídeos/análise , Espectrometria de Massas/métodos , Engenharia Metabólica/métodos , Oligossacarídeos/química , Plasmodium falciparum/química , Cromatografia em Camada Fina , Eritrócitos/química , Corantes Fluorescentes/química , Glicolipídeos/química , Glicoesfingolipídeos/química , Glicosilação , Glicosilfosfatidilinositóis/química , Plasmodium falciparum/patogenicidade , Coloração e RotulagemRESUMO
The cell surface of Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These molecules are involved in a variety of interactions between this parasite and its mammalian and insect hosts. Here, using the neutral detergent Triton X-114, we obtained fractions rich in GPI-anchored and other membrane proteins from insect developmental stages of T. cruzi. These fractions were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS), resulting in the identification of 98 proteins of metacyclic trypomastigotes and 280 of epimastigotes. Of those, approximately 65% (n=245) had predicted lipid post-translational modification sites (i.e., GPI-anchor, myristoylation, or prenylation), signal-anchor sequence, or transmembrane domains that could explain their solubility in detergent solution. The identification of some of these modified proteins was also validated by immunoblotting. We also present evidence that, in contrast to the noninfective proliferative epimastigote forms, the infective nonproliferative metacyclic trypomastigote forms express a large repertoire of surface glycoproteins, such as GP90 and GP82, which are involved in adhesion and invasion of host cells. Taken together, our results unequivocally show stage-specific protein profiles that appear to be related to the biology of each T. cruzi insect-derived developmental form.
Assuntos
Detergentes/farmacologia , Proteômica/métodos , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/métodos , Biologia Computacional/métodos , Glicoproteínas/química , Glicosilfosfatidilinositóis/química , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Octoxinol , Polietilenoglicóis/farmacologia , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
The reconstitution of membrane proteins into liposomes is a useful tool to prepare antigenic components that induce immunity. We have investigated the influence of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol molar ratio on the incorporation of a GPI-protein from Leishmania amazonensis on liposomes and Langmuir monolayers. The latter system is a well behaved and practical model, for understanding the effect of variables such as surface composition and lipid packing on protein incorporation. We have found that the DPPC/cholesterol molar ratio significantly alters the incorporation of the GPI-protein. In the absence of cholesterol, reconstitution is more difficult and proteoliposomes cannot be prepared, which we correlated with disruption of the DPPC layer. Our results provide important information that could be employed in the development of a vaccine system for this disease or be used to produce other GPI-systems for biotechnological application.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Antígenos de Protozoários/química , Materiais Biomiméticos/química , Colesterol/química , Leishmania/química , Proteínas de Membrana/química , Adsorção , Animais , Glicosilfosfatidilinositóis/química , Lipossomos/química , Microdomínios da Membrana/química , Solubilidade , Propriedades de Superfície , Fatores de TempoRESUMO
GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Glicosilfosfatidilinositóis/química , Leishmania braziliensis/imunologia , Proteínas de Membrana , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Biomarcadores , Eletroforese em Gel de Poliacrilamida , Glicosilfosfatidilinositóis/metabolismo , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Proteínas de Membrana/isolamento & purificação , CoelhosRESUMO
GPI-anchored proteins from plasma membrane of Trypanosoma rangeli and Trypanosoma cruzi epimastigotes were isolated and characterized using the partition Triton X-114 method. The detection by Western blot of specific proteins of 90, 85 and 56 kDa molecular mass in T. rangeli compared to those of 30, 70 and 100 kDa detected in T. cruzi demonstrates specific discrimination between these two species of Trypanosoma. The potential diagnostic value of the here reported proteins to differentiate mixed infections by T. cruzi and T. rangeli is evaluated and its potential for epidemiological studies of Chagas disease in endemic areas is also discussed.
Assuntos
Glicosilfosfatidilinositóis/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/química , Animais , Western Blotting , Doença de Chagas/parasitologia , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Glicosilfosfatidilinositóis/química , Humanos , Proteínas de Membrana/química , Peso Molecular , Octoxinol , Polietilenoglicóis , Proteínas de Protozoários/química , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
This work investigates the process of incorporation of a glycosylphosphatidyl inositol (GPI)-anchored alkaline phosphatase into Langmuir monolayers of dimyristoyl phosphatidic acid (DMPA). Three different methods of protein incorporation were assayed. When the protein solution was injected below the air-water interface after formation of the lipid monolayer a micro-heterogeneous distribution of alkaline phosphatase throughout the interface was observed. Adsorption kinetics studied by fluorescence microscopy, associated with surface pressure measurements, led to the proposition of a model in which the protein penetration is modulated by the surface packing of the monolayer and intermolecular interactions occurring between the phospholipid and the protein. At initial surface pressures higher than 20 m Nm(-1), the protein is quickly adsorbed on the interface and the lateral diffusion drives the alkyl chains to turn towards the air phase while the polypeptide moiety faces the aqueous subphase.
Assuntos
Fosfatase Alcalina/química , Glicosilfosfatidilinositóis/química , Membranas Artificiais , Adsorção , Animais , Glicerofosfolipídeos/química , Tamanho da Partícula , Ratos , Propriedades de Superfície , Fatores de TempoRESUMO
The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.
Assuntos
Fosfatase Alcalina/química , Glicosilfosfatidilinositóis/química , Membranas Artificiais , Ar , Fosfatase Alcalina/metabolismo , Sítios de Ligação , Catálise , Elasticidade , Glicosilfosfatidilinositóis/metabolismo , Hidrólise , Microscopia de Fluorescência , Nitrofenóis/química , Nitrofenóis/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Conformação Proteica , Propriedades de SuperfícieRESUMO
The major free glycoinositolphospholipids and protein-linked glycoinositolphospholipids in Trypanosoma cruzi contain ceramide as the lipid moiety. Ceramide was not found in mammalian glycosylphosphatidylinositol (GPI)-anchors. An alkylglycerol, either as a lyso species or acylated has been also found in T. cruzi anchors. However, unlike African trypanosomes, no diacylglycerol was detected in the GPI-anchors. Using a membrane preparation from epimastigotes upon labelling with UDP[3H]GlcNAc we identified [3H]GlcNAcPI as the first step of GPI biosynthesis. Both, alkylacylglycerol (major) and diacylglycerol are constituents of the lipid. Although inositolphosphoceramide is the main inositolphospholipid in epimastigotes, it does not incorporate GlcNAc. The de-N-acetylation step afforded [3H]GlcN(alkylacylglycerol)PI and we also detected the [3H]GlcN(lysoacyl)PI. A new metabolite, phosphoGlcN(lysoacyl)PI, which was formed on long incorporation times, was characterized by chemical and enzymatic degradations. Several [3H]-Man labelled GPI precursors were obtained by in vitro GDP[3H]-Man labelling in the presence of UDPGlcNAc. All of them were sensitive to PI-PLC and to saponification conditions, thus, supporting a glycerolipid structure.
Assuntos
Acetilglucosamina/análogos & derivados , Glicolipídeos/biossíntese , Glicoesfingolipídeos/metabolismo , Fosfolipídeos/biossíntese , Trypanosoma cruzi/metabolismo , Acetilglucosamina/análise , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Animais , Sistema Livre de Células , Cromatografia em Camada Fina , Glicolipídeos/química , Glicosilação , Glicosilfosfatidilinositóis/biossíntese , Glicosilfosfatidilinositóis/química , Metabolismo dos Lipídeos , Lipídeos/química , Manose/metabolismo , Ácido Palmítico/metabolismo , Fosfatidilinositóis/análise , Fosfatidilinositóis/química , Fosfolipídeos/química , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/química , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
In this study we have optimised the enzyme immunoassay (ELISA) to quantify CD59 antigen in human serum or plasma. The glycosyl-phosphatidylinositol (GPI)-linked form of CD59 is known to complex with serum high-density lipoprotein. For ELISA optimisation, therefore, we investigated the effect of detergents, added to the sample diluent, on the determined values of CD59. Values obtained in the presence of octyl-glucoside (OG) for 20 adults aged 18-35 years and 17 children 1-5 years old were, respectively, 33-119 ng/ml (mean +/- S.D.: 66+/-22 ng/ml) and 37-143 ng/ml (76+/-33 ng/ml). These results were higher than those measured without OG and were in contrast with published results showing absence, or eight to nine times lower levels, of the protein in serum. A known range for serum concentrations of CD59 in healthy individuals will establish an important reference point for clinical work and for the investigation of diseases involving the complement membrane attack complex (MAC) and its regulation.
Assuntos
Antígenos CD59/sangue , Antígenos CD59/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Glucosídeos/química , Glicosilfosfatidilinositóis/metabolismo , Adolescente , Adulto , Antígenos CD59/química , Antígenos CD59/imunologia , Pré-Escolar , Glicosilfosfatidilinositóis/química , Humanos , Lactente , Octoxinol , Polietilenoglicóis/químicaRESUMO
The cell surface of the promastigote stage of the protozoan parasite, Leishmania mexicana is coated by a number of glycosylphosphatidylinositol (GPI)-anchored proteins, a GPI-anchored lipophosphoglycan (LPG) and an abundant class of free GPIs, termed glycoinositolphospholipids (GIPLs). We have developed a new screen for isolating L. mexicana mutants that are defective in GPI biosynthesis, involving concanavalin A selection of a parental strain with a modified surface coat. One mutant was isolated that lacked the major GIPL species and mature GPI-protein anchor precursors, but synthesized normal levels of LPG anchor precursors. Based on analysis of apolar GIPLs that accumulate in this mutant and in vivo and in vitro synthesized GPIs, this mutant was found to have a defect in the addition of an alpha1-6 linked mannose to the common precursor, Man(1)GlcN-PI. The apolar GIPLs were transported to the cell surface with the same kinetics as mature GIPLs. However, non-anchored isoforms of the major GPI-anchored protein, gp63, were either slowly secreted (with a t(1/2) of 2 h) or retained within the endoplasmic reticulum, respectively. These findings suggest that common enzymes are involved in the synthesis of GIPLs and protein anchors and have implications for understanding how the biosynthesis of the major surface components of these parasites is regulated.
Assuntos
Glicolipídeos/biossíntese , Glicosilfosfatidilinositóis/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Fosfolipídeos/biossíntese , Proteínas de Protozoários/biossíntese , Animais , Sequência de Carboidratos , Células Cultivadas , Concanavalina A/metabolismo , Glicolipídeos/química , Glicolipídeos/genética , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/química , Estágios do Ciclo de Vida , Manose/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosfolipídeos/química , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
It has been proposed that self and protozoan-derived GPI anchors are natural ligands of CD1d. In this study, we investigated the ability of GPI anchors from Trypanosoma cruzi to bind to CD1d and mediate activation of NKT cells. We observed that GPI-anchored mucin-like glycoproteins (GPI mucins), glycoinositolphospholipids (GIPLs), and their phosphatidylinositol moieties bind to rCD1d and inhibit the stimulation of a NKT hybridoma by the alpha-galactosylceramide-CD1 complex. However, these GPI anchors and related structures were unable to activate NKT cells in vitro or in vivo. We found that high titers of Ab anti-GPI mucins, but not anti-GIPLs, were detected in sera from wild-type as well as in TAP1(-/-), CD1d(-/-), and MHC class II(-/-) mice after immunization. However, T-dependent anti-GPI mucin Ab isotypes, such as IgG1, IgG2a, IgG2b, and IgG3, were absent on MHC class II(-/-), but were conserved in CD1d(-/-) and TAP1(-/-) mice. Furthermore, we found that CD1d(-/-) mice presented a robust cytokine as well as anti-GPI mucins and anti-GIPL Ab responses, upon infection with T. cruzi parasites. These results indicate that, despite binding to CD1d, GPI mucins and related structures expressed by T. cruzi appear not to evoke dominant CD1d-restricted immune responses in vivo. In contrast, MHC class II is critical for the production of the major Ig G isotypes against GPI mucins from T. cruzi parasites.
Assuntos
Antígenos CD1/metabolismo , Glicoproteínas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Células Matadoras Naturais/imunologia , Mucinas/metabolismo , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Trypanosoma cruzi/metabolismo , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Antígenos CD1/biossíntese , Antígenos CD1/genética , Antígenos CD1/fisiologia , Antígenos CD1d , Ligação Competitiva/imunologia , Sequência de Carboidratos , Células Cultivadas , Doença de Chagas/genética , Doença de Chagas/imunologia , Citocinas/biossíntese , Feminino , Predisposição Genética para Doença , Glicoproteínas/fisiologia , Glicosilfosfatidilinositóis/administração & dosagem , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/fisiologia , Imunidade Inata/genética , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/parasitologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mucinas/administração & dosagem , Mucinas/química , Mucinas/fisiologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Transdução de Sinais/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/parasitologia , Trypanosoma cruzi/química , Trypanosoma cruzi/imunologiaRESUMO
Chagas' disease is a chronic disease affecting millions of people in Latin America. The cell surface of Trypanosoma cruzi, the etiological agent, is covered by a glycocalyx whose components play important roles in parasite survival and infectivity. The most abundant surface component is a glycolipid (glycoinositol phospholipid, GIPL) related in structure to glycosylphosphatidyl inositol anchors. In this review, we describe the biological effects of highly purified native GIPLs and their glycan or lipid moities on cells of the host immune system.
Assuntos
Doença de Chagas/imunologia , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/imunologia , Transdução de Sinais , Trypanosoma cruzi/imunologia , Animais , Sequência de Carboidratos , Humanos , Linfócitos/imunologia , Macrófagos/imunologia , Dados de Sequência Molecular , Relação Estrutura-Atividade , Trypanosoma cruzi/fisiologiaRESUMO
A strong activation of macrophages is observed during acute infection with Trypanosoma cruzi. Little is known, however, about the parasite molecules that are responsible for this early activation of innate immunity. Recent studies have shown the stimulatory activity of protozoan-derived glycosylphosphatidylinositol (GPI) anchors on cultured macrophages. In this review, we provide a detailed analysis of the correlation between structure and proinflammatory activity by T. cruzi-derived GPI anchors. We also cover the studies that have identified the Toll-like receptor 2 as a functional GPI receptor and have partially characterized signaling pathways triggered by T. cruzi-derived GPI anchors, which lead to the synthesis of proinflammatory cytokines in macrophages. Finally, we discuss the implications of these findings in resistance and pathogenesis during the infection with T. cruzi.
Assuntos
Doença de Chagas/imunologia , Proteínas de Drosophila , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/fisiologia , Trypanosoma cruzi/patogenicidade , Animais , Sequência de Carboidratos , Citocinas/biossíntese , Inflamação/imunologia , Lipopolissacarídeos/química , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Receptor 2 Toll-Like , Receptores Toll-LikeRESUMO
The most important glycoproteins of trypanosomatids are anchored by glycoinositolphospholipids (GIPLs) to their plasma membrane. In addition, free GIPLs have been described, for instance the lipopeptidophosphoglycan (LPPG) which is a major component of the surface of T. cruzi epimastigotes. An inositolphosphoceramide (IPC) is part of the LPPG and of glycoproteins present in different stages of T. cruzi. Ceramide was not found in mammal GIPL-anchors. The lipid moieties in T. cruzi anchors can be quite variable. However, no diacylglycerol (DAG) was found in contrast with the African trypanosomes. In GIPLs of epimastigotes collected at the logarithmic phase of growth both, 1-O-hexadecyl-2-O-palmitoylglycerol and ceramide were identified. Lignoceroylsphinganine is the major ceramide, however, no lignoceric acid was detected when analysing the candidate precursors IPCs, in any of the stages of T. cruzi. An alkylglycerol has been found either as a lyso species in the Tc85 glycoprotein of trypomastigotes or acylated as in the 1G7 anchor of metacyclic forms and in the mucins of epimastigote forms. The lipid in the mucins is replaced by ceramide when the parasite differentiates to metacyclic forms. Also, in the Ssp-4 glycoprotein characteristic of amastigotes, a ceramide was identified as the anchor lipid. These variations suggest that a remodelling mechanism is working in T. cruzi. On the other hand, the oligosaccharide core in the GIPLs of T. cruzi is substituted with galactofuranose. This monosaccharide is found only in the pyranose configuration in mammalian glycoproteins and glycolipids. Thus, the biosynthetic steps for the introduction of galactofuranose and ceramide in the anchors of T. cruzi are good targets for the development of therapeutic agents.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Antiprotozoários/farmacologia , Sequência de Carboidratos , Desenho de Fármacos , Glicosilfosfatidilinositóis/química , Dados de Sequência Molecular , Proteínas de Protozoários/química , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Glycosylphosphatidylinositol (GPI) anchor and glycoinositolphospholipid (GIPL) are abundant molecules present in the membrane of parasitic protozoa that are common etiologic agents of medical and veterinary diseases. Recent studies have documented the immunostimulatory/regulatory activity of protozoan-derived GPI-anchors and related structures. Among the bioactivity displayed by the protozoan-derived GPI-anchor is the ability to elicit the synthesis of pro-inflammatory cytokines as well as nitric oxide by host macrophages. In contrast, at high concentrations GIPL and lipophosphoglycan (LPG) derived from protozoan parasites suppress several functions of the host immune system. Additionally, the protozoan-derived GPI-anchor and GIPL have been shown to serve as targets for both specific B and NK-T lymphocyte responses. This information extends our knowledge about parasite molecules that stimulate/regulate the host immune system during protozoan infection. The identification of receptor(s) and signaling pathways triggered by these GPI-related glycolipids may provide new insights for the development of therapies that inhibit detrimental immune responses or potentiate beneficial immune responses observed during infection with protozoan parasites.