Full text
| |
| Author(s): |
Jade Cabestre Venancio Brochi
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2022-02-21 |
| Examining board members: |
Ana Patricia Yatsuda Natsui;
Hamilton Cabral
|
| Advisor: | Ana Patricia Yatsuda Natsui |
| Abstract | |
Neospora caninum, an apicomplexan parasite, is the etiological agent of neosporosis, a disease that leads to neurological symptoms in dogs and abortion in cattle. Vaccine or drug treatments for neosporosis remain to be determined. The development of the parasite inside host cells is characterized by the active secretion of proteins, allied to the tight control of the redox status. In this sense, the elucidation of new molecules and mechanisms related to redox control of N. caninum may contribute to the development of neosporosis treatments. Therefore, here it was described two enzymes of redox system: Peroxiredoxin (NcPrx, Chapter 1) and Glutathione reductase (NcGR, Chapter 2), as well it was investigated the effects of the recombinant forms of NcGR and NcPrx, and H2O2 in the tachyzoite invasion and proliferation in Vero cell (Chapter 3). Both NcPrx and NcGR amino-acid sequence showed high identity and similarity compared to homologues representatives of the Apicomplexa phylum. The recombinant NcPrx (rNcPrx) and recombinant NcGR (rNcGR) were cloned and expressed in Escherichia coli (BL21) with the predicted molecular weight (22 kDa and 52kDa, respectively) and the identity of monomer and dimer forms of rNcPrx and rNcGR were confirmed by mass spectrometry. Immunofluorescence analysis showed that NcPrx and NcGR are located in tachyzoite\'s cytosol. Native and recombinant NcPrx and NcGR were detected by ELISA and western blot, using the polyclonal anti-rNcPrx and anti-rNcGR sera. rNcPrx demonstrated peroxidase and antioxidant activity in vitro. Moveover, H2O2 increased the NcPrx dimerization in intracellular and extracellular tachyzoites. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340. rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG): 0.076 ± 0.003 s-1; Km(NADPH): 0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). Furthermore, rNcGR was inhibited by phenothiazinium dyes (IC50 (Methylene blue): 2.1 ± 0.2 µM, IC50(1,9-dimethyl methylene blue): 11 ± 2 µM, IC50(new methylene blue): 0.7 ± 0.1 µM, e IC50(toluidine blue O): 0.9 ± 0.2 µM). In chapter 3, it was demonstrated that rNcPrx interfered in the N. caninum invasion in a redox state manner. Oxidized rNcPrx inhibited the N. caninum invasion and proliferation with no toxic effects observed in Vero cells. In contrast, lower concentrations of H2O2 (10 µM) stimulated the N. caninum invasion, which was reverted in higher doses (> 100 µM). H2O2 inhibited the parasite proliferation in lower concentrations (IC50: 320 µM) compared to the cytotoxicity in host cells (IC50: 586 µM), resulting in a positive selectivity index (1.8). Finally, our results suggest the importance of NcPrx and NcGR in N. caninum biology and antioxidant mechanisms. Data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition and that NcPrx is enrolled in H2O2 clearance and sensing in the parasite. Besides, we showed a connection between the N. caninum development and the redox state, contributing to the elucidation of mechanisms related to parasite propagation and control. (AU) | |
| FAPESP's process: | 19/05758-5 - Redox control in Neospora caninum: investigation of antioxidant system enzymes |
| Grantee: | Jade Cabestre Venancio Brochi |
| Support Opportunities: | Scholarships in Brazil - Doctorate |