RESUMO
The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of vicilin in larval C. maculatus by employing ex vivo experiments. In the ex vivo approach, we incubated FITC-labelled vicilin with isolated midgut wholemounts in the absence or in the presence of endocytosis inhibitors. The fate of labelled or non-labelled globulins was monitored by confocal microscopy and fluorescence measurement. Our results suggest that the internalization of vicilins is due to receptor-mediated endocytosis. Here we report the identity of a microvillar vicilin-binding protein that was purified using affinity chromatography on a vicilin-sepharose column. The putative vicilin receptor showed high homology to proteins with the CRAL-TRIO domain, specifically the Sec14 superfamily member α-tocopherol transfer protein. The precise mechanism involved in vicilin internalization was defined through the use of specific inhibitors of the endocytosis pathway. The inhibitors filipin III and nystatin significantly inhibited the endocytosis of vicilin, while chlorpromazine and phenylarsine oxide had a much lower effect on endocytosis, suggesting that the endocytic pathway is predominantly mediated by caveolin.
Assuntos
Proteínas de Transporte/metabolismo , Besouros/metabolismo , Células Epiteliais/metabolismo , Proteínas de Insetos/metabolismo , Larva/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sequência de Aminoácidos , Animais , Arsenicais/farmacologia , Transporte Biológico , Proteínas de Transporte/genética , Clorpromazina/farmacologia , Besouros/efeitos dos fármacos , Besouros/genética , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/metabolismo , Sistema Digestório/ultraestrutura , Endocitose/efeitos dos fármacos , Endocitose/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Filipina/farmacologia , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Expressão Gênica , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Nistatina/farmacologia , Proteínas de Armazenamento de Sementes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Coloração e RotulagemRESUMO
Caveolin is the protein marker of caveola-mediated endocytosis. Previously, we demonstrated by immunoblotting and immunofluorescence that an anti-chick embryo caveolin-1 monoclonal antibody (mAb) recognizes a protein in amoeba extracts. Nevertheless, the caveolin-1 gene is absent in the Entamoeba histolytica genome database. In this work, the goal was to isolate, identify and characterize the protein that cross-reacts with chick embryo caveolin-1. We identified the protein using a proteomic approach, and the complete gene was cloned and sequenced. The identified protein, E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L), is a member of the StAR-related lipid transfer (START) protein superfamily. The human homolog binds and transfers phosphatidylcholine (PC) and phosphatidylethanolamine (PE) between model membranes in vitro; however, the physiological role of PCTP-L remains elusive. Studies in silico showed that EhPCTP-L has a central START domain and also contains a C-terminal intrinsically disordered region. The anti-rEhPCTP-L antibody demonstrated that EhPCTP-L is found in the plasma membrane and cytosol, which is in agreement with previous reports on the human counterpart. This result points to the plasma membrane as one possible target membrane for EhPCTP-L. Furthermore, assays using filipin and nystatin showed down regulation of EhPCTP-L, in an apparently cholesterol-independent way. Interestingly, EhPCTP-L binds primarily to anionic phospholipids phosphatidylserine (PS) and phosphatidic acid (PA), while its mammalian counterpart HsPCTP-L binds neutral phospholipids PC and PE. The present study provides information that helps reveal the possible function and regulation of PCTP-L expression in the primitive eukaryotic parasite E. histolytica.
Assuntos
Entamoeba histolytica/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Caveolina 1/imunologia , Membrana Celular/metabolismo , Embrião de Galinha , Colesterol/metabolismo , Reações Cruzadas , Citoplasma/metabolismo , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/genética , Filipina/farmacologia , Dados de Sequência Molecular , Nistatina/farmacologia , Fosfatidilcolinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/imunologia , Proteínas de Transferência de Fosfolipídeos/isolamento & purificação , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfoproteínas/química , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologiaRESUMO
Bordetella pertussis is a re-emerging human respiratory pathogen whose infectious process is not fully understood, hampering the design of effective vaccines. The nature of bacterial attachment to host cells is a key event in the outcome of the infection. However, host cell receptors involved in B. pertussis colonization of the respiratory tract are still under investigation. Here, we report that cholesterol-rich domains are involved in B. pertussis adhesion to epithelial cells. Treatment of A549 cells with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin, nystatin, or filipin resulted in a significant decrease of B. pertussis attachment. Confocal laser microscopy studies showed B. pertussis associated with cholesterol-rich domains. Accordingly, B. pertussis was found in detergent-resistant membrane domain fractions isolated from bacterial-infected A549 cells. Our results indicate a main role of filamentous hemagglutinin, an environmentally regulated virulence factor, in this interaction, and a specific affinity for cholesterol, one of the major components of tracheal secretions, which might additionally contribute to the effective colonization of the respiratory tract.
Assuntos
Aderência Bacteriana , Bordetella pertussis/fisiologia , Colesterol/metabolismo , Células Epiteliais/microbiologia , Adesinas Bacterianas/metabolismo , Antimetabólitos/farmacologia , Linhagem Celular , Filipina/farmacologia , Humanos , Nistatina/farmacologia , Fatores de Virulência de Bordetella/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Transferrin uptake by Trypanosoma cruzi epimastigotes occurs mainly through the cytostome/cytopharynx. Here, we present evidences for the association of sterol-rich membrane domains with the transferrin endocytic site. Assays using pharmacological treatments to disrupt clathrin-coated pits and hinder caveolae formation showed no association between transferrin uptake and clathrin-dependent endocytosis, but indicated that cholesterol stability in membrane domains is essential for the endocytosis of transferrin. Furthermore, it was observed a connection between the integrity of cytoskeleton elements at the cytopharynx and the function of the cytostome. Our data show that T. cruzi epimastigotes depend on a specialized pathway for transferrin uptake, which is cholesterol-dependent, clathrin-independent, and closely associated with the structural stability of the cytostome/cytopharynx cytoskeleton.
Assuntos
Colesterol/metabolismo , Clatrina/fisiologia , Citoesqueleto/fisiologia , Endocitose/fisiologia , Transferrina/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Anti-Infecciosos/farmacologia , Citocalasina B/farmacologia , Citoesqueleto/ultraestrutura , Endocitose/efeitos dos fármacos , Filipina/farmacologia , Citometria de Fluxo , Microscopia Eletrônica de Transmissão , Trypanosoma cruzi/ultraestrutura , beta-Ciclodextrinas/farmacologiaRESUMO
Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.
Assuntos
Macrófagos Peritoneais/parasitologia , Microdomínios da Membrana/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Cavéolas/parasitologia , Caveolina 1/análise , Toxina da Cólera/farmacologia , Colesterol/metabolismo , Endocitose , Filipina/farmacologia , Gangliosídeo G(M1)/análise , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/análise , Camundongos , Microscopia Confocal , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-beta-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.
Assuntos
Endocitose , Rotavirus/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cavéolas/metabolismo , Linhagem Celular , Toxina da Cólera/metabolismo , Clatrina/antagonistas & inibidores , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Filipina/farmacologia , Macaca mulatta , Mutação , Nistatina/farmacologiaRESUMO
1. Amphotericin B (Am.B) was shown to have a direct effect on T. cruzi, with the three forms of the parasite presenting different susceptibilities to the drug in the following order: amastigotes > trypomastigotes > epimastigotes. These differences highlight the importance of using the vertebrate forms of the parasite in tests of new drugs. 2. The treated parasites showed alterations of the plasma membrane, suggesting that, as in fungi, the primary effect of Am.B was probably via formation of complexes with membrane components. 3. When exposed to filipin, another polyene antibiotic, the three parasite forms were observed to present a similar order of susceptibility, with comparable ultrastructural modifications. 4. Higher concentrations of Am.B were required to damage the intracellular parasites in vitro, 2.3 micrograms/ml for parasites inside peritoneal macrophages and 7 micrograms/ml for parasites inside heart muscle cells. 5. Am.B is effective against the parasite, but is also toxic to mammalian cells. Testing of Am.B for the control of Chagas' disease by blood transfusion may be useful, since bloodstream forms are lysed by lower concentrations of the drug than those required to affect intracellular parasites.
Assuntos
Anfotericina B/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Transfusão de Sangue , Doença de Chagas/prevenção & controle , Relação Dose-Resposta a Droga , Filipina/farmacologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , CamundongosRESUMO
1. Amphotericin B (Am.B) was shown to have a direct effect on T. cruzi, with the three forms of the parasite presenting different susceptibilities to the drug in the following order: amastigotes > trypomastigotes > epimastigotes. These differences highlight the importance of using the vertebrate forms of the parasite in tests of new drugs. 2. The treated parasites showed alterations of the plasma membrane, suggesting that, as in fungi, the primary effect of Am.B was probably via formation of complexes with membrane components. 3. When exposed to filipin, another polyene antibiotic, the three parasite forms were observed to present a similar order of susceptibility, with comparable ultrastructural modifications. 4. Higher concentrations of Am.B were required to damage the intracellular parasites in vitro, 2.3 micrograms/ml for parasites inside peritoneal macrophages and 7 micrograms/ml for parasites inside heart muscle cells. 5. Am.B is effective against the parasite, but is also toxic to mammalian cells. Testing of Am.B for the control of Chagas' disease by blood transfusion may be useful, since bloodstream forms are lysed by lower concentrations of the drug than those required to affect intracellular parasites
Assuntos
Animais , Anfotericina B/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Transfusão de Sangue , Doença de Chagas/prevenção & controle , Relação Dose-Resposta a Droga , Filipina/farmacologia , Macrófagos Peritoneais , Interações Hospedeiro-ParasitaRESUMO
The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.