Clonagem e caracterização da proteína 14-3-3 de Paracoccidioides brasiliensis durante sua interação com células epiteliais
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2011-11-18
Autores
Silva, Julhiany de Fátima da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A paracoccidioidomicose (PCM) é micose sistêmica causada pelos fungos dimórficos Paracoccidioides brasiliensis (espécies cripticas S1, PS2, PS3) e Paracoccidioides lutzii (Pb01-like espécies), endêmica na América Latina, principalmente no Brasil. A capacidade de causar micose com grande variedade de manifestações clínicas, desde formas localizadas até doença disseminada evoluindo para letalidade, depende da virulência do fungo e de sua habilidade em interagir com as estruturas superficiais do hospedeiro e invadi-las, e a resposta imunológica deste último. P. brasiliensis tem a capacidade de aderir e invadir células epiteliais de linhagens humanas e animais e o processo de invasão do fungo afeta a estrutura do citoesqueleto, interferindo em aspectos morfológicos da actina, tubulina e citoqueratina, indicando a participação funcional dos microfilamentos e microtúbulos neste mecanismo. Em estudos prévios foi identificada adesina de P. brasiliensis de 30 kDa, ligante de laminina, caracterizada como uma proteína pertencente à família 14-3-3. O objetivo deste trabalho foi produzir a proteína recombinante, verificar o papel desta na interação do fungo com células epiteliais pulmonares de linhagem contínua. Para tanto, com o anticorpo anti-14-3-3 foi verificada a sua localização, tanto nas formas leveduriformes do fungo como no processo de interação. A clonagem foi realizada em vetor de clonagem pCR2.1-TOPO - pET32a, para permitir a expressão da proteína Pb14-3-3r com cauda de histidina no espaço periplasmático de E. coli. A fração periplasmática foi purificada por cromatografia de afinidade seguida de eletroeluição. Foi possível verificar alteração do padrão de distribuição de actina quando as células foram tratadas com a proteína recombinante e nativa. A proteína 14-3-3 apresenta distribuição ubíqua na...
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis (cryptic species S1, PS2, PS3) and Paracoccidioides lutzii (Pb01-like species), endemic in Latin America, mainly Brazil. The ability to cause mycosis with a variety of clinical manifestations, from localized forms to disseminated disease progressing to lethality, possibly depends on the relationship between the virulence of these, the ability to interact with the surface structures of the host and invade them, and this immune response. P. brasiliensis has the ability to adhere and o invade human and animal epithelial cells lineage and the process of invasion of the fungus affects the structure of the cytoskeleton, morphological interfering with actin, tubulin and cytokeratin morphology, indicating the functional participation of microfilaments and microtubules in this mechanism. Previous study identified a 14-3-3 protein (30 kDa) that acts as a P. brasiliensis adhesin (laminin ligand) and this protein contributes to the virulence of this important fungal pathogen. The objective of this study was to produce recombinant protein, to verify the role of the interaction of the fungus with lung epithelial cells of continuous lineage. To do so, with the anti-14-3-3 was verified its location, both in the forms of the fungus and yeast in the process of interaction. The cloning was done in cloning vector pCR2.1-TOPO - pET32a to allow expression of protein Pb14-3-3r-tailed histidine periplasmic space of E. coli. The periplasmic fraction was purified by affinity chromatography followed by electroelution. It was possible to see change in the pattern of distribution of actin when cells were treated with recombinant and native protein. The 14-3-3 protein has ubiquitous distribution in yeast cells of P. brasiliensis (strain 18/S1) have a certain... (Complete abstract click electronic access below)
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis (cryptic species S1, PS2, PS3) and Paracoccidioides lutzii (Pb01-like species), endemic in Latin America, mainly Brazil. The ability to cause mycosis with a variety of clinical manifestations, from localized forms to disseminated disease progressing to lethality, possibly depends on the relationship between the virulence of these, the ability to interact with the surface structures of the host and invade them, and this immune response. P. brasiliensis has the ability to adhere and o invade human and animal epithelial cells lineage and the process of invasion of the fungus affects the structure of the cytoskeleton, morphological interfering with actin, tubulin and cytokeratin morphology, indicating the functional participation of microfilaments and microtubules in this mechanism. Previous study identified a 14-3-3 protein (30 kDa) that acts as a P. brasiliensis adhesin (laminin ligand) and this protein contributes to the virulence of this important fungal pathogen. The objective of this study was to produce recombinant protein, to verify the role of the interaction of the fungus with lung epithelial cells of continuous lineage. To do so, with the anti-14-3-3 was verified its location, both in the forms of the fungus and yeast in the process of interaction. The cloning was done in cloning vector pCR2.1-TOPO - pET32a to allow expression of protein Pb14-3-3r-tailed histidine periplasmic space of E. coli. The periplasmic fraction was purified by affinity chromatography followed by electroelution. It was possible to see change in the pattern of distribution of actin when cells were treated with recombinant and native protein. The 14-3-3 protein has ubiquitous distribution in yeast cells of P. brasiliensis (strain 18/S1) have a certain... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Paracoccidioidomicose, Paracoccidioides brasiliensis, Micoses fungoides, Celulas epiteliais, Mycosis fungoides
Como citar
SILVA, Julhiany de Fátima da. Clonagem e caracterização da proteína 14-3-3 de Paracoccidioides brasiliensis durante sua interação com células epiteliais. 2011. 133 f. Tese (doutorado) - Universidade Estadual Paulista Julio de Mesquita Filho, Faculdade de Ciências Farmacêuticas, 2011.