RESUMO
Trypanosoma cruzi can modulate a large number of host intracellular responses during its invasion. GTPases such as RhoA, Rac1 and Cdc42 are examples of molecules that could be activated at this moment and trigger changes in the pattern of F-actin cytoskeleton leading to the formation of structures like stress fibers, lamellipodium and fillopodium, respectively. Here we investigate the role of these GTPases in the cytoskeletal rearrangement of MDCK cell transfectants expressing variants of RhoA, Rac1 and Cdc42 during T. cruzi infection. The adhesion, internalization and the survival rate were determined. Rac1 mutants showed the higher adhesion and internalization indexes but the lower survival index after 48 h of infection. Confocal laser scanning microscopy showed changes in the pattern of F-actin distribution and reorganization at the site of trypomastigote invasion. These observations suggest that these GTPases act in the signaling mechanisms that affect the F-actin cytoskeleton during T. cruzi invasion.
Assuntos
GTP Fosfo-Hidrolases/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Actinas/análise , Animais , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Cães , Microscopia Confocal , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
One fundamental step of Leishmania-macrophage interaction is the phase of parasite internalization through an endocytic process, with the formation of the parasitophorous vacuole (PV). The present study analyzed this process using two approaches. First, to investigate the host cell proteins which take part in this compartment, the macrophage surface was biotinilated and allowed to interact with both Leishmania forms, the PV was then isolated, and the biotinilated proteins were analyzed by Western blot. The results obtained showed that the isolated PV from macrophages infected for 60 min with infective promastigotes displayed high molecular weight proteins, 220 kDa and 180 kDa, contrary to the isolated PV obtained from amastigotes. The isolated PV from amastigotes, after 60 min interaction, displayed a faint, biotinilated protein profile, in contrast to the PV containing amastigote which, after 30 min interaction, displayed a strong protein profile in the range of 120 kDa and 40-60 kDa. The biotinilated protein profile may represent proteins distributed in the PV membrane and may also correspond to biotinilated proteins incorporated by the intracellular parasite, as observed by confocal microscopy. In a second approach, to investigate the PV phospholipid composition, macrophages were incubated with (32)P, allowed to interact with the parasites, and the isolated PV was then processed for phospholipid analysis by thin layer chromatography and scintillation counting. An increase in the levels of lysophosphatidylcholine was observed in infected macrophages. The isolated PV from infective promastigotes and amastigotes, after 60 min interaction, displayed high levels of phosphatidylcholine. Then the PV was ruptured and the intravacuolar parasite's (32)P phospholipid composition was analyzed by TLC; and labeling of the parasites was found, suggesting that phospholipids from the macrophage are transferred to the parasite. Taken together, the results obtained show that several proteins and phospholipids found in the plasma membrane of the macrophage are also found in the PV compartment.
Assuntos
Leishmania/fisiologia , Macrófagos/parasitologia , Proteínas de Membrana/análise , Fosfolipídeos/análise , Vacúolos/parasitologia , Animais , Membrana Celular/metabolismo , Membrana Celular/parasitologia , Cricetinae , Interações Hospedeiro-Parasita , Leishmania/classificação , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Vacúolos/química , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
In the present study the parental cells and glycosylation mutants of Chinese hamster ovary (CHO) cells were used to analyze the influence of surface carbohydrates on the cytoadhesion of trichomonads. Trichomonas vaginalis and Tritrichomonas foetus were allowed to interact with host cells for 2 h at 37 degrees C. Alternatively, CHO cells were treated with 10 mM periodate prior to the assays. Both trichomonads adhered to all CHO cell clones tested. A remarkable difference could be observed between the cytoadhesion of T. vaginalis and T. foetus. Sialic acid residues present on the surface of CHO cells may favor the cytoadhesion of T. foetus while hampering that of T. vaginalis. The specificity of the parasite cytoadhesion was further investigated. Sialic acid, mannose, and galactose as well as mannose, galactose, and N-acetylglucosamine added to the interaction medium at 50, 100, and 200 mM were capable of significantly inhibiting the cytoadhesion of each trichomonad species. Periodate treatment of target cells also induced decreases in the cytoadhesion of the trichomonads. These results strongly suggest an important role for host-cell surface glycoconjugates during the cytoadhesion of trichomonads. In addition, they also point out the presence of "lectin-like" molecules on the surface of both T. vaginalis and T. foetus.
Assuntos
Adesão Celular , Glicoconjugados/fisiologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Monossacarídeos/farmacologia , Trichomonas vaginalis/fisiologia , Tritrichomonas foetus/fisiologia , Animais , Células CHO , Adesão Celular/efeitos dos fármacos , Membrana Celular/parasitologia , Membrana Celular/ultraestrutura , Células Clonais , Cricetinae , Glicosilação , Microscopia Eletrônica de Varredura , Trichomonas vaginalis/ultraestrutura , Tritrichomonas foetus/ultraestruturaRESUMO
The effect of nanomolar concentrations of 12-O-tetradecanoilphorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus was evaluated by means of measurements of the parasites' surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites' net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.
Assuntos
Acetato de Tetradecanoilforbol/farmacologia , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas/efeitos dos fármacos , Testes de Aglutinação , Animais , Adesão Celular/fisiologia , Oligossacarídeos/análise , Solubilidade , Propriedades de Superfície , Água/químicaRESUMO
1. To further characterize the basic aspects of parasite-cell interaction, untreated and hormone-treated Madin-Darby canine kidney (MDCK) cells were allowed to interact with T. vaginalis. 2. Among the six hormones assayed, alpha-estradiol caused MDCK cells to become highly adhesive to T. vaginalis. Increases in parasite adhesion of 83% and 68% were obtained by previous incubation of the epithelial monolayers for 18 h with 1 ng/ml alpha-estradiol and 10 ng/ml 17-beta-estradiol, respectively. 3. The estrogenic response of MDCK cells was inhibited by tamoxifen, cycloheximide, puromycin, or 2-deoxy-D-glucose. 4. We conclude that MDCK cells possess receptors for estrogens and that T. vaginalis cytoadherence can be influenced by the hormonal cycle of its host.
Assuntos
Adesão Celular/efeitos dos fármacos , Estrogênios/farmacologia , Trichomonas vaginalis/efeitos dos fármacos , Animais , Células Cultivadas , Cães , Epitélio/efeitos dos fármacos , Epitélio/parasitologia , Técnicas In Vitro , Rim/citologia , Microscopia Eletrônica , Fatores de TempoRESUMO
The feeding behavior of six triatomid species toward latex condoms filled with blood at 26 ñ 1 and 36 ñ 1 grade C observed for 4 h. The species studied were Triatoma infestans, Triatoma brasiliensis, Tritoma vitiiticeps, Triatoma pseudomaculata, Rhodnius and Panstrongylus, and 34 to 348 insects were studied in each group (average, 190). All the life stages of each species were used. Although most insects (80%, average for 6 species) preferred the warmer blood, 54% of the individuals of all species fed on blood kept at 26 grade C. For Triatoma pseudomaculata, a higher proportion of insects fed on blood at 26 grade C (92%) than on blood at 36 grade C (65%). These results suggest that bait containing insecticides which are effective through the digest tract may be useful in the control of these vectors even at room temperature