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- ItemSomente MetadadadosCharacterization of a 21 kDa protein from Trypanosoma cruzi associated with mammalian cell invasion(Elsevier B.V., 2009-04-01) Silva, Claudio V. da [UNIFESP]; Kawashita, Silvia Y. [UNIFESP]; Probst, Christian M.; Dallagiovanna, Bruno; Cruz, Mario C. [UNIFESP]; Silva, Erika A. da [UNIFESP]; Souto-Padron, Thais C. B. S.; Krieger, Marco A.; Goldenberg, Samuel; Briones, Marcelo R. S. [UNIFESP]; Andrews, Norma W.; Mortara, Renato A. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Fiocruz MS; Universidade Federal do Rio de Janeiro (UFRJ); Yale UnivTrypanosoma cruzi genomic database was screened for hypothetical proteins that showed high probability of being secreted or membrane anchored and thus, likely involved in host-cell invasion. A sequence that codes for a 21 kDa protein that showed high probability of being secreted was selected. After cloning this protein sequence, the results showed that it was a ubiquitous protein and secreted by extracellular amastigotes. the recombinant form (P21-His(6)) adhered to HeLa cells in a dose-dependent manner. Pretreatment of host cells with P21-His(6) inhibited cell invasion by extracellular amastigotes from G and CL strains. On the other hand, when the protein was added to host cells at the same time as amastigotes, an increase in cell invasion was observed. Host-cell pretreatment with P21-His(6) augmented invasion by metacyclic trypomastigotes. Moreover, polyclonal antibody anti-P21 inhibited invasion only by extracellular amastigotes and metacyclic trypomastigotes from G strain. These results suggested that P21 might be involved in T. cruzi cell invasion. We hypothesize that P21 could be secreted in the juxtaposition parasite-host cell and triggers signaling events yet unknown that lead to parasite internalization. (C) 2009 Elsevier Masson SAS. All rights reserved.
- ItemAcesso aberto (Open Access)Characterization of the infective properties of a new genetic group of Trypanosoma cruzi associated with bats(Elsevier B.V., 2011-12-01) Maeda, Fernando Yukio [UNIFESP]; Alves, Renan Melatto [UNIFESP]; Cortez, Cristian [UNIFESP]; Lima, Fabio Mitsuo [UNIFESP]; Yoshida, Nobuko [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently described. Here we characterized a T. cruzi strain from this new genetic group, which could be a potential source of infection to humans. Metacyclic trypomastigotes (MT) of this strain, herein designated BAT, were compared to MT of well characterized CL and G strains, as regards the surface profile and infectivity toward human epithelial HeLa cells. BAT strain MT expressed gp82, the surface molecule recognized by monoclonal antibody 3F6 and known to promote CL strain invasion by inducing lysosomal exocytosis, as well as mucin-like molecules, but lacked gp90, which functions as a negative regulator of invasion in G strain. A set of experiments indicated that BAT strain internalization is gp82-mediated, and requires the activation of host cell phosphatidylinositol 3-kinase, protein kinase C and the mammalian target of rapamycin. MT of BAT strain were able to migrate through a gastric mucin layer, a property associated with p82 and relevant for oral infection. Gp82 was found to be a highly conserved molecule. Analysis of the BAT strain gp82 domain, containing the cell binding- and gastric mucin-binding sites, showed 91 and 93% sequence identity with G and CL strains, respectively. Hela cell invasion by BAT strain MT was inhibited by purified mucin-like molecules, which were shown to affect lysosome exocytosis required for MT internalization. Although MT of BAT strain infected host cells in vitro, they were less effective than G or CL strains in infecting mice either orally or intraperitoneally. (C) 2011 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosMammalian cell invasion by closely related Trypanosoma species T. dionisii and T. cruzi(Elsevier B.V., 2012-02-01) Maeda, Fernando Yukio; Cortez, Cristian; Alves, Renan Melatto; Yoshida, Nobuko [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Protozoan parasites of the genus Trypanosoma can infect virtually all mammalian species. Within this genus, Trypanosoma dionisii from bats and Trypanosoma cruzi that causes Chagas' disease, belonging to the subgenus Schizotrypanum, can invade mammalian cells. the mechanisms of cell invasion by T. dionisii are poorly understood. To address that question, metacyclic trypomastigotes (MT) and human epithelial HeLa cells were used. Similarly to genetically divergent T. cruzi strains G (TcI) and CL (TcVI), associated, respectively with marsupial and human infections. T. dionisii infectivity increased under nutritional stress, a condition that induces host cell lysosome exocytosis required for parasite internalization. for efficient internalization, T. dionisii depended on MT protein tyrosine kinase (FTK) and Ca2+ mobilization from acidocalcisomes, whereas T. cruzi strains also relied on phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC) and Ca2+ released from thapsigargin-sensitive compartments. T. dionisii-induced signaling in host cells implicated PKC and Ca2+ mobilized from thapsigargin-sensitive stores, like T. cruzi, but without PI3K involvement. Unlike T. cruzi, T. dionisii metacyclic forms did not use L-proline as source of energy required for internalization. Molecules related to T. cruzi surface glycoproteins involved in MT-host cell interaction were undetectable in T. dionisii. the difference in the surface profile of the two species was also inferred from the susceptibility of T. dionisii metacyclic forms to complement-mediated lysis, as opposed to complete resistance of T. cruzi. in summary, the two Trypanosoma species display distinct surface profiles but invade host cells through a common mechanism involving lysosome mobilization to the site of parasite entry. (C) 2011 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Participação das proteínas ezrina, radixina e moesina (ERM proteins) na invasão celular por amastigotas extracelulares de Trypanosoma cruzi(Universidade Federal de São Paulo (UNIFESP), 2016-11-30) Ferreira, Éden Ramalho de Araujo [UNIFESP]; Mortara, Renato Arruda [UNIFESP]; http://lattes.cnpq.br/3754467086294573; http://lattes.cnpq.br/0533784588961610; Universidade Federal de São Paulo (UNIFESP)The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas? disease that affects 6-7 million people worldwide, mostly in the South and Central America. Chagas? disease was responsible for 76% of all deaths caused by Neglected Tropical Diseases in Brazil from 2000 to 2011. In mammalian hosts T. cruzi alternates from extracellular (infective) trypomastigote forms and intracellular (replicative) amastigote forms. Additionally, trypomastigotes can differentiate into amastigotes in the extracellular environment generating infective extracellular amastigotes (EAs). Host cell invasion by these forms is mediated by complex cellular signaling events regulating actin filaments, the main component in EA uptake. ERM proteins (ezrin, radixin and moesin) are key elements linking actin filaments to the plasma membrane, important for the maintenance of cell morphology, cell migration and invasion of intracellular pathogens. The aim of this study was to evaluate the role of ERM proteins in actin cytoskeleton-plasma membrane interplay during host cell invasion by EAs. Our results revealed that depletion of host ezrin and radixin but not moesin inhibited EAs invasion in HeLa cells. Confocal microscopy of HeLa cells transfected with ERM proteins?GFP or -HA revealed recruitment of ERM proteins to EAs invasion sites colocalizing with F-actin. Additionally, invasion assays performed with cells overexpressing ERM proteins revealed increased EAs invasion in ezrin and radixin but not moesin overexpressing cells. Finally, time-lapse live imaging confocal microscopy has shown reduced and delayed actin dynamics in ezrin and radixin depleted HeLa cells when compared to control or moesin groups. Altogether, these findings show distinct roles of ERM proteins in actin filament dynamics and plasma membrane interplay during EAs host cell invasion.
- ItemSomente MetadadadosRemoval of sialic acid from mucin-like surface molecules of Trypanosoma cruzi metacyclic trypomastigotes enhances parasite-host cell interaction(Elsevier B.V., 1997-01-01) Yoshida, Nobuko [UNIFESP]; Dorta, Miriam Leandro [UNIFESP]; Ferreira, Alice Teixeira [UNIFESP]; Oshiro, Maria Etsuko Miyamoto [UNIFESP]; Mortara, Renato Arruda [UNIFESP]; Serrano, Alvaro Acosta; Favoreto Junior, Silvio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The 35/50 kDa mucin-like surface glycoprotein (gp 35/50) of Trypanosoma cruzi metacycliic trypomastigotes has been in mammalian cell invasion. in this study we investigated whether the sialyl residues of gp35/50 are required for interaction of parasites with target cells. After treatment with bacterial neuraminidase, the metacyclic forms (G strain) remained reactive with the monoclonal antibody (mAb) 10D8 but lost their reactivity with mAb 3C9, that recognizes sialic acid-containing epitopes on gp35/50, and entered HeLa cells in significantly higher numbers as compared to untreated controls. Resialylation of gp35/50, by incubation of parasites with T. cruzi trans-sialidase and sialyl lactose, restored the reactivity with mAb 3C9 as well as the affinity for sialic acid specific lectin. Accordingly, the rate of invasion of resialylated parasites was reduced to levels similar to those observed before desialylation. Purified G strain gp35/50, desialylated by neuraminidase treatment, bound to HeLa cells more than its sialylated counterpart. the Ca2+ signaling activity, which has been associated with cell invasion, was also determined by measuring the cytosolic Ca2+ concentration ([Ca2+](i)), in HeLa cells upon interaction with sonicated extracts from untreated or neuraminidase-treated parasites, or with purified gp35/50 in its sialylated or desialylated form. Consistent with the results of cell invasion assay, the desialylated parasite preparations, as well as the sialic acid free gp35/50, induced an average elevation in [Ca2+](i) significantly higher than that triggered by untreated controls. None of these effects, namely the increase in infectivity and Ca2+ signaling activity, was observed with neuraminidase-treated CL strain metacyclic trypomastigotes, which express a variant form of sialic acid gp35/50 molecule that is not recognized by mAb 10D8 and apparently is not involved in target cell invasion. Copyright (C) 1997 Elsevier Science B.V.
- ItemSomente MetadadadosSerotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli strains isolated from healthy dairy cattle(Wiley-Blackwell, 2016) Gonzalez, Alice Gonçalves Martins; Cerqueira, Aloysio de Mello Figueiredo; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Coutinho, Cesar Augusto de Souza; Liberal, Maira Halfen Teixeira; Souza, Rossiane de Moura; Andrade, João Ramos da Costa; Universidade Federal de São Paulo (UNIFESP)AimThe occurrence of virulence markers, serotypes and invasive ability were investigated in Shiga toxin-producing Escherichia coli (STEC) isolated from faecal samples of healthy dairy cattle at Rio de Janeiro State, Brazil. Methods and ResultsFrom 1562 stx-positive faecal samples, 105 STEC strains were isolated by immuno-magnetic separation (IMS) or plating onto MacConkey agar (MC) followed by colony hybridisation. Fifty (476%) strains belonged to nine serotypes (O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2 and ONT:H21). The prevalent serotypes were O157:H7 (124%), O113:H21 (67%) and O8:H19 (57%). Virulence genes were identified by polymerase chain reaction (PCR). E-hlyA (771%) was the more prevalent virulence marker, followed by espP (648%), saa (39%), eae (248%) and astA (219%). All O157:H7 strains carried the (gamma) variant of the locus of enterocyte effacement (LEE) genes and the stx2c gene, while the stx1/stx2 genotype prevailed among the eae-negative strains. None of the eae-positive STEC produced the localized adherence (LA) phenotype in HEp-2 or Caco-2 cells. However, intimate attachment (judged by the fluorescent actin staining test) was detected in some eae-positive strains, both in HEp-2 (231%) and in Caco-2 cells (115%). Most strains (875%) showed peripheral association' (PA) adherence phenotype to undifferentiated Caco-2 cells. Twenty-five (926%) of 27 strains invaded Caco-2 cells. The highest average value of invasion (96%) was observed among the eae-negative bovine strains from serotypes described in human disease. ConclusionHealthy dairy cattle is a reservoir of STEC carrying virulence genes and properties associated with human disease. Significance and Impact of the StudyAlthough reports of human disease associated with STEC are scarce in Brazil, the colonization of the animal reservoir by potentially pathogenic strains offers a significant risk to our population.
- ItemSomente MetadadadosUnique behavior of Trypanosoma dionisii interacting with mammalian cells: Invasion, intracellular growth, and nuclear localization(Elsevier B.V., 2009-04-01) Castro Oliveira, Miriam Pires de [UNIFESP]; Cortez, Mauro [UNIFESP]; Maeda, Fernando Yukio [UNIFESP]; Fernandes, Maria Cecilia [UNIFESP]; Haapalainen, Edna Freymuller [UNIFESP]; Yoshida, Nobuko [UNIFESP]; Mortara, Renato Arruda [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The phylogenetic proximity between Trypanosoma cruzi and Trypanosoma (Schizotrypanum) dionisii suggests that these parasites might explore similar strategies to complete their life cycles. T, cruzi is the etiological agent of the life threatening Chagas' disease, whereas T dionisii is a bat trypanosome and probably not capable of infecting humans. Here we sought to compare mammalian cell invasion and intracellular traffic of both trypanosomes and determine the differences and similarities in this process. the results presented demonstrate that T dionisii is highly infective in vitro, particularly when the infection process occurs without serum and that the invasion is similarly affected by agents known to interfere with T. cruzi invasion process. Our results indicate that the formation of lysosomal-enriched compartments is part of a cell-invasion mechanism retained by related trypanosomatids, and that residence and further escape from a lysosomal compartment may be a common requisite for successful infection. During intracellular growth, parasites share a few epitopes with T. cruzi amastigotes and trypomastigotes. Unexpectedly, in heavily infected cells, amastigotes and trypomastigotes were found inside the host cell nucleus. These findings suggest that T. dionisii although sharing some features in host cell invasion with T. cruzi, has unique behaviors that deserve to be further explored. (c) 2009 Elsevier B.V. All rights reserved.