Caracterização molecular da proteína histidina quinase de Paracoccidioides brasiliensis

• Autor(a) principal: Santos, Dener Lucas Araújo dos
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/9457
• topic: Paracoccidioides spp.; Histidina quinase; Expressão heteróloga; Paracoccidioides spp.; Histidine kinase; Heterologous expression; GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

Prokaryotic and eukaryotic organisms (except those belonging to the phylum metazoa) have developed a system named phosphorelay, which is responsible for adaptation to different environmental conditions such as oxidative stress, osmotic stress and thermal shock. Studies have demonstrated that inhibition of hybrid histidine kinase, the stimulus sensory protein in the phosphorelay pathway, affects morphological transition from mycelial fungi to yeast. Among the thermodymorphic fungi, Paracoccidioides spp. are among the most prevalent in Latin America, mainly in Brazil, as the cause of the systemic mycosis paracoccidioidomycosis (PCM). The exposure of fungi from this complex to the host body temperature (37 ° C), leads to the transition from the mycelial to the parasitic yeast form. It is notable that the morphological transition is necessary for the pathogenicity of Paracoccidioides spp.. Thus, the objective of this study is to characterize the hybrid histidine kinase (HHK) protein and to evaluate its level of expression during the dimorphism of Paracoccidioides brasiliensis. We carried out similarity analyzes that indicated that HHK is highly conserved in other dimorphic fungi such as H. capsulatum and B. dermatitidis. In addition, a region of the HHK gene was cloned into pET32a expression vector and the recombinant protein was generated. After confirming the identity of HHK in UPLC-MSE, we produced polyclonal anti-histidine kinase antibodies. RT-qPCR revealed that PbHHK is expressed in greater amounts transition when compared to the other morphological phases. In conclusion, the high conservation of histidine kinase in dimorphic fungi combined with their increased expression in the transition phase demonstrates the importance of PbHHK for the morphological transition of P. brasiliensis.