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Redox control in Neospora caninum: investigation of antioxidant system enzymes

Grant number: 19/05758-5
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): June 01, 2019
Effective date (End): May 31, 2020
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Ana Patricia Yatsuda Natsui
Grantee:Jade Cabestre Venancio Brochi
Home Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The apicomplexan protozoan Neospora caninum (Nc), the etiological agent of Neosporosis, is associated with neurological symptoms in dogs and abortion in cattle. This disease is worldwide spread and has no efficient treatment. The survival and replication of the parasite depends on an enzymatic antioxidant system defense for evading the oxidative stress inside the host cell. Among these enzymes, thioredoxin-dependent peroxide reductase (NcPrx) and glutathione reductase (NcGR) draw our attention due to a significant relative abundance in the proteome of Neospora caninum, allied to the absence of literature. Therefore, we will perform a characterization of the N. caninum antioxidant enzymes NcPrx and NcGR to elucidate their role in the successful parasite establishment and proliferation in the host cell. The NcGR and NcPrx recombinant proteins (rNcGR and rNcPrx) will be cloned and expressed followed by SDS-PAGE characterization and mass spectrometry identification. Through polyclonal antibodies production, NcGR and NcPrx will be detected in the parasite by immunofluorescence and western blot. The activity of the recombinant enzymes will be evaluated by specific enzymatic assays and their biochemical behavior against phenothiazine dyes and conoidin A will be tested. In addition, the action of NcPrx and NcGR in the response of N. caninum to oxidative stress will be demonstrated by the profile of antioxidant enzymes expressed after H2O2 stimulation and employing recombinant NcPrx and NcGR enzymes in tachyzoite proliferation assays in H2O2 presence. Ultimately, this project will reveal novel features of fundamental enzymes of the N. caninum redox system enabling investigations of drugs with unknown mechanisms of action and advances on the research of Neosporosis. (AU)