RESUMO
Extracellular vesicles, whether microvesicles (MVs) or exosomes, shed by pathogens transfer virulence factors and biomolecules to host cells, thereby altering the host's susceptibility to infection. We have previously demonstrated that MV release is increased during the interaction between the infective forms of Trypanosoma cruzi and host cells. MVs confer parasite resistance to complement-mediated lysis and enhance parasite invasion. In this study, we show that differences exist in the levels of MVs released during the interaction between metacyclic trypomastigotes of different T. cruzi strains (with varied sensitivity to complement-mediated lysis, namely sensitive G strain TcI and resistant Y strain TcII) and host cells. MVs produced during the interaction between TcII parasites and host cells increased parasite resistance to complement lysis from 50% to 80% and parasite invasion was increased to over 50%. MVs purified during the interaction between TcI parasites and host cells have a stronger effect, doubling complement resistance and parasite invasion. The complement-mediated lysis assays showed that all MVs inhibit mainly the lectin pathway. Interestingly, MVs derived from parasites of one class did not alter complement resistance and the invasion process of parasites from the other class. This is the first description of MVs from T. cruzi with strain-dependent phenotypic effects.
Assuntos
Via Alternativa do Complemento , Proteínas do Sistema Complemento/genética , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Parasita , Trypanosoma cruzi/patogenicidade , Animais , Sobrevivência Celular , Chlorocebus aethiops , Vesículas Extracelulares/imunologia , Expressão Gênica , Humanos , Transdução de Sinais , Especificidade da Espécie , Células THP-1 , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/imunologia , Células Vero , VirulênciaRESUMO
Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell-derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP-1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell-derived trypomastigote (tissue culture-derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP-1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP-1-derived MVs can fuse with parasite-derived MVs. Furthermore, MVs derived from the TCT-THP-1 interaction showed a higher fusogenic capacity than those from META- or EPI-THP-1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite-THP-1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP-1-derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Doença de Chagas/parasitologia , Monócitos/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Antígenos de Protozoários/imunologia , Micropartículas Derivadas de Células/parasitologia , Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Chlorocebus aethiops , Interações Hospedeiro-Parasita , Humanos , Fusão de Membrana , Camundongos Endogâmicos BALB C , Monócitos/metabolismo , Proteoma/metabolismo , Células VeroRESUMO
Community-based small-scale reforestation practices have been proposed as an alternative to low-efficiency massive reforestations conducted by external agents. These latter conventional reforestations are often carried out in soils that have been seriously degraded and this has indirectly contributed to the introduction of non-native species and/or acceptance of very low seedling survival rates. Bokashi is a fermented soil organic amendment that can be made from almost any available agricultural byproduct, and its beneficial effects in agriculture have been reported in various contexts. Here, we report the results of a community-based small-scale experimental reforestation where the provenance of pine seedlings (local and commercial) and the use of Bokashi as a soil amendment were evaluated. Bokashi was prepared locally by members of a small rural community in central Mexico. Almost two years after the establishment of the trial, survival rates for the unamended and amended local trees were 97-100% while survival of the commercial trees from unamended and amended treatments were 87-93%. Consistently through time, local and commercial seedlings planted in Bokashi-amended soils were significantly taller (xÌ = 152 cm) than those planted in unamended soils (Ì x = 86 cm). An unplanned infection by Cronartium quercuum in the first year of the experiment was considered as a covariable. Infected seedlings showed malformations but this did not affect survival and growth rates. Bokashi amendment seems as an inexpensive, locally viable technology to increase seedling survival and growth and to help recover deforested areas where soils have been degraded. This allows local stakeholders to see more rapid results while helping them to maintain their interest in conservation activities.
Assuntos
Fertilizantes/análise , Agricultura Florestal , Pinus/crescimento & desenvolvimento , Solo/química , Humanos , México , Plântula/crescimento & desenvolvimentoRESUMO
Protease expression among TCI and TCII field isolates was analysed. Gelatin-containing gels revealed hydrolysis bands with molecular masses ranging from 45 to 66 kDa. The general protease expression profile showed that TCII isolates presented higher heterogeneity compared to TCI. By utilizing protease inhibitors, we showed that all active proteases at acid pH are cysteine-proteases and all proteases active at alkaline pH are metalloproteases. However, the expression of cruzipain, the T. cruzi major cysteine-protease, did not reproduce a heterogeneous TCII cysteine zymogram profile. Dendogram analyses based on presence/absence matrices of proteases and cruzipain bands showed a TCI separation from the TCII group with 50-60% similarity. We suggest that the observed cysteine protease diversification contributes to differential host infection between TCI and II genotypes.
Assuntos
Cisteína Endopeptidases/genética , Peptídeo Hidrolases/genética , Trypanosoma cruzi/enzimologia , Animais , Western Blotting , Cisteína Endopeptidases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genótipo , Interações Hospedeiro-Parasita/genética , Filogenia , Inibidores de Proteases/farmacologia , Proteínas de Protozoários , Trypanosoma cruzi/genéticaRESUMO
Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5% and 9% with IHA and ELISA tests, respectively, and up to 7.7% using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7%), Bolivia (13.7%) and Argentina (3.5%). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1%) and AIDS (0.1%) in the same region.
Assuntos
Anticorpos Antiprotozoários/sangue , Doadores de Sangue , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Adolescente , Adulto , Idoso , Animais , Doença de Chagas/diagnóstico , Doença de Chagas/epidemiologia , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Humanos , México/epidemiologia , Pessoa de Meia-Idade , Prevalência , Estudos SoroepidemiológicosRESUMO
Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5 percent and 9 percent with IHA and ELISA tests, respectively, and up to 7.7 percent using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7 percent), Bolivia (13.7 percent) and Argentina (3.5 percent). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1 percent) and AIDS (0.1 percent) in the same region
Assuntos
Animais , Humanos , Adolescente , Adulto , Pessoa de Meia-Idade , Doadores de Sangue , Doença de Chagas , Trypanosoma cruzi , Anticorpos Antiprotozoários , Doença de Chagas , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , México , Prevalência , Estudos Soroepidemiológicos , Trypanosoma cruziAssuntos
Proteínas Luminescentes/genética , Transfecção , Trypanosoma cruzi/genética , Animais , Antibacterianos/farmacologia , Meios de Cultura , Eletroporação , Fluorescência , Amplificação de Genes , Expressão Gênica , Vetores Genéticos , Gentamicinas/farmacologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde , Neuraminidase/genética , Neuraminidase/metabolismo , Plasmídeos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. In contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T. cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.
Assuntos
Glicoproteínas de Membrana/genética , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Clonagem Molecular , Cães , Glicosilação , Mamíferos , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Transfecção , Células VeroRESUMO
We describe the effect of an inositol phosphoglycan (IPG) purified from Trypanosoma cruzi on the stimulation of aldosterone and cAMP production by ACTH in calf adrenocortical cells. T. cruzi IPG has two galactofuranose residues (Galf) which are not frequent in other IPGs. The effect of IPG with galactofuranose residues (IPG Galf) and IPG without these residues (IPG) was investigated. It was found that IPG Galf slightly decreased the stimulation of aldosterone and cAMP production by ACTH, whereas IPG significantly inhibited ACTH-mediated accumulation of both aldosterone and cAMP. The inhibition of aldosterone content in ACTH-treated cells by IPG was dose dependent. It was also found that the pretreatment of calf adrenocortical cells with IPG inhibited the accumulation of aldosterone provoked by ACTH and dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP). On the other hand, the activation of a GPI (glycosyl phosphatidylinositol)-phospholipase C by ACTH was evaluated. First it was found that the release of ceramide from a GPI-like molecule: a glycoinositol-phosphoceramide (LPPG) purified from T. cruzi is increased in ACTH-treated cells. Second, the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium was increased in these cells by ACTH. These data suggest that ACTH activates a phospholipase C in calf adrenocortical cells, releasing IPG, which in turn may inhibit, or modulate ACTH action.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/antagonistas & inibidores , Antagonistas de Hormônios/farmacologia , Fosfatos de Inositol/farmacologia , Polissacarídeos/farmacologia , Trypanosoma cruzi/química , Córtex Suprarrenal/citologia , Córtex Suprarrenal/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Aldosterona/biossíntese , Fosfatase Alcalina/metabolismo , Animais , Bucladesina/farmacologia , Sequência de Carboidratos , Bovinos , Células Cultivadas , AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Ativação Enzimática , Glicoesfingolipídeos/química , Antagonistas de Hormônios/isolamento & purificação , Fosfatos de Inositol/química , Fosfatos de Inositol/isolamento & purificação , Dados de Sequência Molecular , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/metabolismo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Fosfolipases Tipo C/metabolismoRESUMO
Between 1988-1922, data of the nutritional status of pregnant women seen in the Santiago Metropolitan Health Service were analyzed. Underweight (22.2%), normal weight (47.2%), overweight (19.7%) and Obese (15.4%). Four thousand five hundred fifty five pregnant women were studied. Underweight 1136, normal weight 1219, overweight 1100 and obese 1100. Underweight was significantly more frequent in the patients less than 20 years old while overweight and obese was significantly more frequent in the patients over 30 years old. Hypertension (2.6%) was the only significant morbidity factor in the obese group. The overweight and obese groups had earlier menarche, while the obese group had shorter periods. The obese group were associated most frequently with higher parity (75.1%), stillbirth (4.6%), spontaneous abortion (19.5%), induced abortion (3.1%) and high obstetric risk (33.2%). In the normogram used, the underweight patients are abnormally represented at the start of pregnancy. The obese group gained less weight proportionally during pregnancy (overweight and obese 42.8%, underweight and normal 34.7%). The obese group presented more frequently with hypertension (20.4%) and diabetes (0.7%), while the obstetric complications occurred more frequently in the underweight (6.3%). The underwent group had more anemia (45.4%) and premature labor (12.3%). Cesarean section was performed more frequently in the obese group (33.1% versus 21.3% of all the other groups combined. The neonatal birthweight was in direct proportion to the maternal weight, measured by various methods. It is worth noting the importance of microelements in the milk ingestion of the pregnant patients and the influence on their weight.
Assuntos
Peso Corporal , Estado Nutricional , Gravidez , Aborto Espontâneo/etiologia , Adolescente , Adulto , Anemia/complicações , Índice de Apgar , Peso ao Nascer , Glicemia/análise , Aleitamento Materno , Criança , Dieta , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Ciclo Menstrual , Necessidades Nutricionais , Paridade , Estudos Retrospectivos , Fatores SocioeconômicosRESUMO
The lipopeptidophosphoglycan from Trypanosoma cruzi is a glycosylated inositol-phosphoceramide isolated from epimastigotes at the stationary phase of growth (4-5 days). We have now purified two similar glycoinositolphospholipids (glycoinositolphospholipid A and glycoinositolphospholipid B) from epimastigotes after the second day of culture growth. [3H]Palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in glycoinositolphospholipid A and into ceramide in glycoinositolphospholipid B. The lipids were released by incubation with glycosylphosphatidylinositol-specific phospholipase C from Bacillus thuringiensis or by chemical methods. After alkaline hydrolysis, the lipids were analysed by GLC/MS. In glycoinositolphospholipid A the resulting lipids corresponded to 1-O-hexadecylglycerol and palmitic acid. The ceramide components in glycoinositolphospholipid B are sphinganine, palmitic acid and lignoceric acid. The oligosaccharides could be degraded by nitrous acid and further enzymic treatment showed that the two glycoinositolphospholipids isolated from T. cruzi share the common core structure of the glycosylphosphatidylinositol membrane anchors. The microheterogeneity was determined, as well as the substitution by galactose, and was mainly in the furanose configuration as was previously described for lipopeptidophosphoglycan. However, methylation analysis indicated that 20% of the galactose is in the pyranose form. Both glycoinositolphospholipids mainly differ in the lipid moiety.
Assuntos
Ceramidas/química , Éteres de Glicerila/química , Glicosilfosfatidilinositóis/química , Palmitatos/química , Trypanosoma cruzi/química , Animais , Sequência de Carboidratos , Cromatografia Gasosa , Glicosilação , Glicosilfosfatidilinositóis/isolamento & purificação , Lipídeos/análise , Espectrometria de Massas , Metilação , Dados de Sequência Molecular , Polissacarídeos/análise , Polissacarídeos/química , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
This study provides several pieces of evidence indicating that 3F6-Ag, identified by monoclonal antibody (MAb) 3F6 as a stage-specific glycoprotein of approximately 82 kDa on the surface of metacyclic trypomastigotes of different Trypanosoma cruzi strains, promotes the entry of parasites into host cells through a ligand-receptor type interaction. First, invasion of Vero cells by metacyclic trypomastigotes of both CL and Tulahuen strains was significantly inhibited by MAb 3F6 or its Fab fragments. Second, purified 3F6-Ag bound to Vero cells in a dose-dependent and saturable fashion. Third, soluble 3F6-Ag reduced the infection of Vero cells by metacyclic forms of CL and Tulahuen strains by 90 to 97 and 50%, respectively. Unrelated proteins, as well as extracellular matrix components, such as heparan sulfate and collagen, had no effect. Our studies also show that in the Tulahuen strain, 10D8-Ag, a 35/50-kDa glycoprotein identified by MAb 10D8, participates in target cell invasion, confirming previous observations, but the variant form of 10D8-Ag expressed by highly invasive CL strain metacyclic trypomastigotes appears to be irrelevant. Overall, our results indicate that the surface components of T. cruzi metacyclic trypomastigotes involved in the process of host cell penetration are developmentally regulated molecules, such as 3F6-Ag and 10D8-Ag, that have no counterpart in blood- or tissue culture-derived trypomastigotes.
Assuntos
Antígenos de Protozoários/fisiologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/patogenicidade , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/imunologia , Glicoproteínas de Membrana/fisiologia , Trypanosoma cruzi/fisiologia , Células VeroRESUMO
The lipopeptidophosphoglycan is the major cell surface glycoconjugate of the epimastigote forms of the parasitic protozoan Trypanosoma cruzi. A detailed partial structure for this molecule has been reported (Previato, J. O., Gorin, P. A. J., Mazurek, M., Xavier, M. T., Fournet, B., Wieruszesk, J. M., and Mendonca-Previato, L. (1990) J. Biol. Chem. 265, 2518-2526). In this study, we complete the primary structure assignments and describe the microheterogeneity found in the lipopeptidophosphoglycan glycan, using a combination of 1H and 31P NMR, fast atom bombardment mass spectrometry, methylation linkage analysis, and exoglycosidase sequencing. The lipopeptidophosphoglycan is a glycosylated inositol-phosphoceramide with striking homology to glycosylphosphatidylinositol membrane anchors found attached to a wide variety of plasma membrane proteins throughout the eukaryotes.
Assuntos
Oligossacarídeos/química , Peptidoglicano/química , Fosfolipídeos/química , Trypanosoma cruzi/química , Acilação , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Peptidoglicano/isolamento & purificação , Fosfolipídeos/isolamento & purificação , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol-specific phospholipase C (PtdIns-specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non-N-acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry. The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns-specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 micrograms carbohydrate caused 60% inhibition of the binding of anti-CRD antibodies raised against the soluble form of variant surface glycoprotein.
Assuntos
Glicolipídeos/análise , Peptidoglicano/isolamento & purificação , Fosfatidilinositóis/análise , Fosfolipídeos/isolamento & purificação , Trypanosoma cruzi/análise , Animais , Ceramidas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Glicosilfosfatidilinositóis , Oxirredução , Peptidoglicano/análise , Fosfolipídeos/análise , Fosfolipases Tipo CRESUMO
The presence and the importance of a preovulatory prolactin (PRL) peak was determined in four, natural or artificially induced, ovulatory models related to lactation in the rat. Gonadotrophin peaks were determined in the afternoon preceding ovulation in four models: postpartum ovulation (PPO), ovulation after the lactational period (AL) (natural models), ovulation after litter removal at midlactation (ML), and ovulation in lactating rats (LR) (artificially induced models). In PPO, AL, and ML rats a preovulatory PRL surge was detected, showing that its presence is a common characteristic of ovulation in the rat. Bromocriptine inhibition of PRL levels in PPO and AL rats did not modify the percentage of rats which ovulated. In contrast, this treatment was able to significantly increase ovulation percentage in ML rats. Moreover, in LR rats strong dopaminergic inhibition of PRL levels, induced by pergolide, was necessary for ovulation to take place, but if pergolide-treated rats were injected with ovine PRL ovulation was completely inhibited. These data suggest that while a PRL surge seems to be always present in natural ovulatory models, it is not essential for ovulation to take place. On the other hand, in artificially induced ovulatory models, suppression of prolactinemia is able to induce ovulation or to increase the percentage of rats which ovulated. This effect of PRL on ovulation may be direct or indirect.