Validation of the functional importance of natterins in the innate immune response in zebrafish by CRISPR-Cas9, a loss-of-function method

Abstract

The family of Natterins, found in the venom of the fish Thalassophryne nattereri, has five orthologs (1 - 4 and -P) with N-terminal (DM9) and C-terminal domains (aerolysin). Natterin-like protein sequences can be found in different immune defense cells of various species of non-venomous and venomous fish; yet recent studies have shown the participation of Natterin-like proteins in the immune defense against pathogens. Aerolysin-like proteins, known as pore-forming proteins (PFTs), are present in approximately 30% of all protein/toxins in pathogenic bacteria and are capable of activating the NRLP3 inflammasome. Through of bioinformatic analyzes, some aerolysin isoforms have been found in the zebrafish model organism that share a 60% identical sequence to the Natterin-like proteins of catfish and lamprey, which are hypothetically considered as defense molecules against predators. Zebrafish has proven to be an excellent model organism for genetic manipulation because of its transparency, temporal separation of immune responses, easy and low cost maintenance and manipulation, and its system enables high resolution quantitative images, as well as having 70% homology with the human gene. Our group has proven in previous work that the poisoning by Natterins in mice induces the recruitment of inflammatory mediators and inflammatory cells to the site of the injured tissue. To test the hypothesis that Natterins act as defense molecules in host fish, we will use zebrafish as a model organism that expresses genes coding for Natterin-like proteins; and through the CRISPR-Cas9 knockdown system, one of the most efficient loss-of-function methods, we will evaluate the homeostatic and pathogen function of Natterins. (AU)