Validação da importância funcional das Natterinas na resposta imune inata em zebrafish por CRISPR/Cas9, um método de loss-of-function

Abstract

Zebrafish presents Natterin-like isoforms that share identity with the Natterina family, an important aerolysin for membrane penetration and inflammasome activation. The aim of this project is to silence the natterin-like gene in zebrafish using the CRISPR/Cas9 depletion system to assess the molecule's action in the body's immune defense against pathogens and its homeostatic function. Initially, we verified the susceptibility of zebrafish to infection with pathogens that activate the canonical and non-canonical pathways of the inflammasome. Thus, zebrafish larvaes with 1 day post fertilization (dpf) were exposed for 2 h to the bacteria Staphylococcus aureus and to Salmonella typhimurium for analysis of the effect on survival, embryonic development, locomotor activity and activation of the inflammasome immediately after the infection. The larvaes showed defects in embryonic development and behavioral change, and responded to infection by S. aureus and S. typhimurium with the production of mature caspase-a. Then, after synthesizing the sgRNA complex (trcarRNA + natterin-like crRNA, XM_017356964 / 2) for the complementary target in the second exon of the natterin-like gene on chromosome 7 (ID: 795232) the complex was microinjected into 0 embryos hpf with Cas9 enzyme in different concentrations (50 μg / nL: 250 μg / nL, 50: 125, 25: 125 and 12.5: 125) for gene depletion and obtaining KO embryos. The complete absence of natterin-like in the entire body of embryos depleted by CRISPR/Cas9 was confirmed by in situ hybridization. All concentrations delayed embryonic development with changes in the body's morphology with anomalies development and induced persistent mortality up to 144 hpf, dose-dependent. Lastly, WT larvae and depleted larvae of the natterin-like gene by CRISPR/Cas9 (sgRNA 50 ng/μL: Cas9 250 ng/μL) were exposed to infection in 1 dpf by S. aureus and S. typhimurium for 15 min, 30 min, 1 h and 2 h. Immediately after infection, the effect on survival, embryonic development, locomotor activity and activation of the inflammasome was evaluated. The absence of natterin-like in larvae (1 dpf) altered the response to infection by S. aureus and S. typhimurium inducing changes in locomotor activity in zebrafish associated with the decreased production of caspase-a and

caspase-b at all times of infection performed. Our results show that natterin-like is crucial for the embryonic development of zebrafish and acts as an effector molecule in the innate immune response against bacterial pathogens activating the inflammasome complex in zebrafish.