Redox signalling and innate immunity: a role for protein S-nitrosylation in the immune response of Drosophila melanogaster
Homem, Rafael Augusto
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Over the past three decades, nitric oxide (NO) has been recognised as one of the most versatile and important players in many aspects of physiology, including immune responses. More recently, S-nitrosylation, the incorporation of a NO moiety into a protein thiol group, has emerged as a major post-translational modification (PTM) during pathophysiological responses in plants and animals. The main goal of this work was to investigate the role of S-nitrosylation in physiology and innate immunity of animals using the genetic reference system, Drosophila melanogaster. The S-nitrosylated derivative of glutathione (GSH), S-nitrosoglutathione (GSNO), is the main non-protein S-nitrosothiol (SNO) in the cell and extracellular fluids. GSNO can trans-S-nitrosylate other thiols and is considered a reservoir of NO bioactivity. The levels of GSNO and total S-nitrosylation have been shown to be controlled by S-nitrosoglutathione reductase (GSNOR) in yeast, plants and mammals. By employing an overlapping deletion technique to knock-out gsnor, a role for S-nitrosylation in the immune response of D. melanogaster is proposed. Compared to wild type flies, gsnor overlapping deletion flies presented lower expression of antimicrobial peptides in response to infections, and succumbed more rapidly to both Gram-positive bacterial and fungal pathogens. As the Toll pathway mediates responses against these pathogens, key components of this network were tested for their propensity to being S-nitrosylated. Two CLIP-domain serine proteases of the Toll signalling pathway, Persephone (PSH) and Spätzle-Processing Enzyme (SPE), were shown to be S-nitrosylated both in vitro and in vivo and this process seemed to control the quaternary structure of these proteins and interfere with the immune response of D. melanogaster. At least for PSH, S-nitrosylation at C254 has an immune significance as the expression of non-Snitrosylable PSHC254S in gsnor knock-out flies partially recovered the resistance of these animals to infections with the entomopathogenic fungus Beauveria bassiana. These findings might represent a novel mechanism by which NO and S-nitrosylation regulate immunity. Further results presented in this thesis reveal an interplay between reactive oxygen species (ROS) and reactive nitrogen species (RNS) in D. melanogaster physiology and immunity. Similarly to what has been reported in Arabidopsis thaliana, gsnor knock-out flies presented higher tolerance to the herbicide paraquat, an inducer of superoxide (O2 -) production. Moreover, additional mutations in Catalase (Cat), a hydrogen peroxide (H2O2) scavenger enzyme, partially restored the immunodeficiency phenotypes of gsnor knock-out flies. These findings suggest an inter-relation between the levels of ROS and RNS during stress responses of plants and animals. In addition, CRISPR/Cas9 technology was employed to generate gsnor knock-outs in the genome of D. melanogaster. These flies were shown to have no GSNOR activity, presented lower tolerance to pharmacological-induced nitrosative stress and succumbed faster to infections with B. bassiana compared to wild type flies. These results support the role played by GSNOR in regulating NO homeostasis and immunity in D. melanogaster.