Programa de Pós-graduação em Genética e Biologia Molecular
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Navegando Programa de Pós-graduação em Genética e Biologia Molecular por Assunto "Adesinas"
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Item Expressão heterológa e imunolocalização das proteínas HSP30 e catalase peroxissomal identificadas na parede de Paracoccidioides spp. interagindo com macrófagos(Universidade Federal de Goiás, 2018-03-09) Pereira, Christie Ataides; Tomazett, Mariana Vieira; http://lattes.cnpq.br/1754626527596461; Soares, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637; Dias, Fátima Ribeiro; Paccez, Juliano Domiraci; Soares, Célia Maria de AlmeidaThe genus Paracoccidioides comprises thermodymorphic ascomycete’s fungi, causative agents of Paracoccidioidomycosis (PCM). PCM is an endemic granulomatous systemic mycosis in Latin America. The pathogen ability to interact and adhere to host surface structures is critical to the colonization, invasion, growth, and hematogenous spread of the fungus to tissues. Fungi use a variety of surface molecules to bind to the components of the host's extracellular matrix and defense cells, such as macrophages, so they can survive in these environments. A total of 94 cell wall proteins of Paracoccidioides spp. interacting with macrophages were identified through mass spectrometry studies. In this sense it becomes important the production of those possible adhesins via heterologous expression and localization of these proteins, aiming to perform adhesion studies. Therefore, HSP30 and peroxisomal catalase proteins of Paracoccidioides brasiliensis were expressed in a bacterial heterologous system, Escherichia coli. Open reading frames (ORFs) of the genes encoding HSP30 and peroxisomal catalase were cloned into pGEX-4T3 expression vector and the respective clones were used in the transformation of E. coli pLySs cells. The recombinant proteins were used in the production of polyclonal antibodies in mice. Anti-HSP30 and anti-CatP polyclonal antibodies were used in immunofluorescence assays, to obtain confirmation of the cellular location of HSP30 and CatP proteins. The obtained data allowed the localization of those proteins in the cell wall of the fungi cells, corroborating with the proteomic analyzes. The aim of this work is to perform additional macrophage interaction experiments to evaluate the potential of both proteins as adhesins.Item Estudo da utilização de heme por Paracoccidioides lutzii: análise do proteoma de parede celular após exposição à hemoglobina e expressão heteróloga de Pga7, um provável receptor de hemoglobina(Universidade Federal de Goiás, 2017-02-21) Souza, Aparecido Ferreira de; Paccez, Juliano Domiraci; http://lattes.cnpq.br/2350706025601982; Soares, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637; Soares, Célia Maria de Almeida; Oliveira, Rosely Maria Zancopé; Bailão, Mirelle Garcia SilvaIron (Fe) is an indispensable metal for most biological systems and is a target of competition in host-pathogen interactions. In humans, most Fe is complexed to the cofactor heme, present in hemoglobin, a molecule that is exploited by pathogens as an iron source. Paracoccidioides spp., a complex of thermodymorphic pathogenic fungi, are the etiological agents of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. Paracoccidioides spp. are able to use hemoglobin in a receptor(PbRbt5)-mediated process. However experimental evidence points to the existence of a complex system with the presence of other proteins. In the present work, we demonstrated the similarity between PAAG_02225 (PbPga7), from Paracoccidioides lutzii, and the sequence of the heme/hemoglobin receptor Pga7 from Candida albicans, and expressed PbPga7 in Escherichia coli. Due to the presence of rare codons in P. lutzii sequence, compared to the codons preferably used by Escherichia coli, chemical synthesis of the gene was employed. The expression of PbPga7 protein opens perspectives for PbPga7 characterization. In addition, nanoUPLC-MSE was employed to analyze P. lutzii cell wall proteome. We observed that the treatment of fungus with hemoglobin promotes induction of potential adhesins and defense-related enzymes against reactive oxygen species. These data indicate that these proteins may be important for the pathogen to access hemoglobin by adhering and lysing erythrocytes, besides counteracting the toxicity generated by heme/hemoglobin released from erythrocytes, allowing the uptake and use of these molecules. The results obtained in the present work reinforce the complexity of the interaction event between pathogen and host and, in addition, contribute to broaden the understanding of the biology of Paracoccidioides spp.