Characterization of an Aspergillus fumigatus PMR1 calcium ATPase.

Author(s)

Frederico Marianetti Soriani

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2006-09-05

Examining board members

Sergio Akira Uyemura; Maria Cristina Roque Antunes Barreira; Lucia Helena Faccioli; Joao Atilio Jorge; Claudia Maria Leite Maffei

• Advisor: Sergio Akira Uyemura

Abstract

The knowledge about the regulation of Aspergillus fumigatus calcium and manganese levels are very limited, while these ions homeostasis could be directly controlled by the function of specific ATPases, like the PMR1 calcium ATPase. In this way, the aim of the present work was the expression, characterization e validation, as chemotherapeutic target, of the A. fumigatus Afpmr1 gene. Initially, the functional complementation of a PMR1 knock-out strain phenotype was analyzed in EGTA or manganese supplemented culture media. Besides, after Afpmr1 expression, an intense distribution of chitin through the cell wall of the knock-out strain was reversed. At the same time, a fragment of the Afpmr1 gene, showing low identity values for another calcium ATPase genes, was cloned in an A. fumigatus expression vector (pALB1) for RNAi. After the induction of gene expression, a double strand RNA construct for RNAi has properly silenced either the alb1 gene alone (control clone), or the double silencing with the gene of interest Afpmr1, leading to both constructions white colored colonies. After confirmation of the gene silencing by quantitative RT-PCR techniques, the selected clones were used in macrophages killing and phagocytosis assays. The Afpmr1 silenced clone showed a decrease in the phagocytosis percentage, in the mean number of internalized conidia and in the killing percentage when compared with control groups. These results show that the Afpmr1 gene can be functionally expressed in eukaryotic heterologous systems and its silencing, in A. fumigatus, alters cellular processes that can be related with the maintenance of the cell wall structure and composition, as well as promote alterations in the macrophages phagocytosis and killing. (AU)