Identification, deletion and characterization of a peroxiredoxin in the human opportunistic pathogen aspergillus fumigatus.

• Grant number: 11/23596-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): February 01, 2012
• Effective date (End): December 31, 2012
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Iran Malavazi
• Grantee: Mayra Mara Ferrari Barbosa
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil

Abstract

During the past 20 years, the incidence of fungal infections in humans has increased considerably. Aspergillus fumigatus is saprophytic filamentous fungus, and the most prevalent fungal pathogen responsible for human respiratory diseases such as pulmonary aspergillosis and allergic bronchopulmonary aspergillosis . In immunocompromised individuals (such as transplant recipients and patients with immunodeficiencies) this pathogen is responsible for invasive pulmonary aspergillosis infections which accounts for high mortality rates. Overall microbial infections trigger an immune response in the host mediated by reactive oxygen species (ROS), which include organic peroxides and hydrogen peroxide. The ability of pathogenic microorganisms to adapt to the host is partially due to the development of mechanisms to protect them against oxidative stress and respiratory burst. Among these mechanisms, is a series of antioxidant enzymes for the detoxification of peroxides which include enzymes catalase, glutathione peroxidase (GPx) and peroxiredoxins (PRX). In this context, the peroxiredoxins has great relevance in the maintenance of the pathogen within the host, by being able to decompose lipid hydroperoxides, peroxynitrite, and hydrogen peroxide. All Prx contain a conserved cysteine residue that participates in a cycle for peroxide-dependent oxidation, thiol-dependent for the reduction during catalysis. The classification is based on the number of cysteines involved in catalysis (1-Cys Prx and 2-Cys Prx) and the type of disulfide that is formed during catalysis: inter-or intramolecular (2-Cys Prx typical or atypical). Analysis of the genome of A. fumigatus using Blastx revealed three open reading frame (ORFs) that exhibit identity with human peroxiredoxina II: Afu4g08580 (33%), Afu5g15070 (29%) and Afu8g07130 (55%). Analysis of the translated ORFs suggests that these are potential 1 Cys Prx (Afu4g08580 and Afu5g15070) and 2-Cys Prx (Afu8g07130) i.e., with two cysteines involved in the catalytic cycle. Interestingly, the 2-Cys Prx typical peroxidase cysteine occupies position closer to the N-terminal (40-50) and, in most cases, is embedded in a motif consisting of Cys-Val-Pro (VCP). However, Afu4g08580 and Afu5g15070 ORFs in spite of presenting similar position in the CP 2-Cys Prx typical CP is inserted in a motif Val-Cys-Glu-Tre-Tre (VCTTE), which is characteristic of prx-1Cys. In particular, the specific case of Prx Afu5g15070, the focus of studies in this work, preliminary biochemical validation experiments developed by our group, strengthen the observation that Afu5g15070 is a 1-Cys prx since it is able to use ascorbate as electrons donor. This project represents a first approach to characterize the system Prx A. fumigatus. Thus, this project aims to assess the biological role of the peroxiredoxin Afu5g15070. To achieve this goal, a null mutant strain for this gene will be isolated in this study. Additionally, the role of the peroxiredoxins in clearance of ROS in A.fumigatus will be traced through the transcriptional profile of the three putative peroxiredoxinas found in the genome of A. fumigatus using real time RT-PCR when a wild type strain was grown in the presence of different agents that generate oxidative stress in the cell, e.g. H2O2, organic peroxides (tert-butyl peroxide), paraquat (methyl viologen) and menadione.