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Construction of a loss-of-function mutant and partial characterization of the hsfA transcription factor in the pathogenic fungus Aspergillus fumigatus

Grant number: 15/18137-8
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): October 01, 2015
Effective date (End): September 30, 2016
Field of knowledge:Biological Sciences - Microbiology
Principal Investigator:Iran Malavazi
Grantee:João Henrique Tadini Marilhano Fabri
Home Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil

Abstract

Aspergillus fumigatus is an opportunistic pathogenic filamentous fungus and the causative agente of several human respiratory diseases, including pulmonary invasive aspergillosis, the life-threatening formo f infection. The thermotolerance are among the major determinants of virulence of this fungus and it is important for the establishment and maintenance of the pathogen inside the host during the infection. The thermotolerance confers allows the fungus to grow at elevated temperatures (i. e. 37°C, in the of mammalian host), thus causing deep mycoses. In Saccharomyces cerevisiae, the transcription factor HSF1 is responsible for the transcription of "heat shock protein", such as the chaperone Hsp90, Hsp70, Hsp60 and Hsp40, which help the cell to survive high temperatures. Yeast HSF1 also promotes the activation of genes encoding enzymes involved in the oxidative stress response, signal transduction, metabolism of carbohydrates, among others. Furthermore, there is also a well-described role between the function of the heat shock genes and the maintenance of the cell wall integrity in fungi, such as in S. cerevisiae and C. albicans. However, the function of the putative HSF1 orthologue gene in A. fumigatus (here, hsfA) and its relationship with the thermotolerance and the cell wall integrity maintenance in this fungal pathogen is still unknown. For this reason, this project aims to expand the study of heat shock response in A. fumigatus through the functional characterization of HsfA transcription factor. hsfA loss- of-function mutants (i.e., null mutant and/or conditional mutant) and a mutant where hsfA will be fused to the GFP protein will be constructed. These mutants will be used in phenotypic tests. The hsfA expression will also be evaluated under different conditions of cell wall stress and high temperature by real time RT-PCR and western blot. The results should contribute to the expansion of knowledge about the mechanisms that regulate the thermotolerance A. fumigatus.