Análise da expressão de genes envolvidos na manutenção da homeostase de cobre no patógeno humano Paracoccidioides brasiliensis

Abstract

The fungus Paracoccidioides brasiliensis, a human pathogen widely distributed in Latin America, causes paracoccidioidomycosis when its mycelia reaches the host lungs. The success of this infection depends on the acquisition of essential micronutrients such as copper, which is required as a cofactor by a variety of enzymes that are important in essential biological processes, such as respiration, growth, and iron acquisition. Previous studies had shown that a high affinity copper transporter (PbCTR3) is a highly expressed molecule that is probably necessary for the establishment of P. brasiliensis infection. In the present study we isolated and characterized the genomic and cDNA sequences coding for PbCTR3 of this pathogen. The cDNA presents 582 base pairs and codes for a protein with 193 amino acids, with predicted molecular mass of 21.5 kDa and pI 8.6. The genomic sequence presents four exons interrupted by three introns. In silico analyses were performed in the database of the structural genome of P. brasiliensis, in which genes involved in the maintenance of copper homeostasis were identified and used to design a model of the factors involved in this process in P. brasiliensis. The transcriptional behavior of the gene Pbctr3 and the genes involved in the maintenance of copper homeostasis were analyzed by real time qRT-PCR during exposure of yeast cells of P. brasiliensis to copper and iron depletion. We demonstrated a significant change in the transcription level of the genes in the absence of copper as well as in the absence of both metals. qRT-PCR was used to analyze, at different times, the expression of Pbctr3 and Pbcrp, which codes a protein responsive to copper, in yeast cells of P. brasiliensis derived from infected lungs and spleen. The expression of Pbctr3 and Pbcrp was super-regulated during the experimental infection. Taken together, these results suggest the importance of PbCtr3 and the system of copper/ iron absorption during the infection process of this pathogen.