Expressão heterológa e imunolocalização das proteínas HSP30 e catalase peroxissomal identificadas na parede de Paracoccidioides spp. interagindo com macrófagos
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2018-03-09
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Universidade Federal de Goiás
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The 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.
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PEREIRA, C. A. Expressão heterológa e imunolocalização das proteínas HSP30 e catalase peroxissomal identificadas na parede de Paracoccidioides spp. interagindo com macrófagos. 2018. 67 f. Dissertação (Mestrado em Genética e Biologia Molecular) - Universidade Federal de Goiás, Goiânia, 2018.