Functional and structural characterization of AfPrxA, a thiol peroxidase from the human pathogen fungus Aspergillus fumigatus and a potential target for drug development

• Grant number: 13/14955-2
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: November 01, 2013
• End date: July 01, 2018
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Luis Eduardo Soares Netto
• Grantee: Renata Bannitz Fernandes
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated scholarship(s): 16/12248-5 - Identification and characterization of acidic Ca2+-independent phospholipase A2 (aiPLA2) activity of three peroxiredoxins from human opportunistic pathogen Aspergillus fumigatus, BE.EP.DD

Abstract

Aspergillus fumigatus is a saprophyte filamentous fungal ubiquitous in the environment and responsible for respiratory human diseases like invasive aspergillosis (AIP), specifically in immunocompromised individuals accounting for high mortality rates. One of the earliest events in the host defense response against pathogen attack is the production of Reactive Oxygen Species (ROS) including H2O2 and organic peroxides. Pathogens possess a number of antioxidant enzymes to peroxide detoxification, which includes catalases, Glutathione Peroxidases (GPx) and Peroxiredoxins (Prx). Additionally to H2O2, the Prx are able to decompose lipid hydroperoxides and peroxynitrite and are considered very important for the survival of the pathogens in the host. Prx uses very reactive cysteines (peroxidatic cysteine-CP) to peroxide decomposition that are present in the universal motif Pro-X-X-X-Thr/Ser-X-X-Cys. This Thr/Ser and Cys, together with a conserved Arg, constitute the Prx catalytic triad, which is involved in the transition-state stabilization of a SN2 nucleophilic substituition reaction. The Prx can be classified according to the number of Cys involved in the catalytic mechanism 1-Cys Prx or 2-Cys Prx (one or two Cys, respectively). AfPrxA is a 1-Cys Prx from Aspergillus fumigatus that possesses high second order constant for H2O2 decomposition (3×107M-1.s-1). In silico analyses reveal that AfPrxA has unique structural features, including substitutions in the motif to Ser-X-X-X-His-X-X-Cys. In order to elucidate its catalytic mechanism, enzymatic assays with the His and Ser mutants and crystallizations with AfPrxA pre-teated with oxidants and reduction agents will be performed. Additionally, the AfPrxA crystallographic structure can be used in future studies of drug design once AfPrxA has low percentage of identity with human Prx counterpart (41%). We also want to express the gene AfPrxA in Saccharomyces cerevisiae. Once we have a collection of yeast mutants and the phenotype of various peroxiredoxin mutants are already characterized, it is possible to analyze if the expression of AfPrxA in one yeast mutant will reverts the yeast wild phenotype. Moreover, tests to elucidate if AfPrxA is involved in the virulence of A. fumigatus be carried out by infection of J774 macrophages with wild strains, AfPrxA or AfPrxAC28S, and analyzing comparatively the death of the macrophages, the secretion level of TNF-± and macrophages oxidative state. We also intend to use the A. fumigatus wild, AfPrxA or AfPrxAC28S strains to evaluate the pathogenicity of AfPrxA in mice. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SANTOS, CARLA SANTANA; BANNITZ-FERNANDES, RENATA; LIMA, ALEX S.; TAIRUM, CARLOS A.; MALAVAZI, IRAN; NETTO, LUIS E. S.; BERTOTTI, MAURO. Monitoring H2O2 inside Aspergillus fumigatus with an Integrated Microelectrode: The Role of Peroxiredoxin Protein Prx1. Analytical Chemistry, v. 90, n. 4, p. 2587-2593, FEB 20 2018. (13/14955-2, 15/02095-4, 15/20776-9, 14/22396-6)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

FERNANDES, Renata Bannitz. Biochemical and structural characterization of peroxiredoxins from Aspergillus fumigatus, human opportunistic pathogen. 2019. Doctoral Thesis - Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) São Paulo.