Enhancing Edwardsiella piscicida resistance through CRISPR/Cas9-mediated deletion of toll-like receptor 5a (tlr5a) in zebrafish.

Abstract

Toll-like receptor 5 (TLR5), a pattern recognition receptor that detects bacterial flagellin, plays a critical role in innate immune responses. Teleost fish possess two paralogs, tlr5a and tlr5b, of which the functional role of tlr5a during flagellated bacterial infections remains unclear. In this study, we investigated the involvement of tlr5a in the immune response against Edwardsiella piscicida infection in zebrafish (Danio rerio). To this end, we generated tlr5a-deficient (tlr5a) zebrafish using CRISPR/Cas9 technology. Survival analysis following E. piscicida challenge revealed significantly higher survival rates and reduced bacterial loads in tlr5alarvae and adults compared to wild-type (WT) controls. tlr5afish exhibited significantly lower expression of pro-inflammatory cytokines (tnfa, il6, and il-1β), chemokine (il8), and pathway genes (nfκb, myd88, and mapk14a) at multiple time points post-infection (0, 6, 12, 24, 48, and 72 hpi). DCFH-DA staining and Sudan Black/Neutral Red staining revealed elevated reactive oxygen species (ROS) levels and immune cell recruitment, respectively, indicating reduced ROS production and diminished neutrophil and macrophage infiltration in tlr5alarvae. Antioxidant gene expression analysis revealed reduced levels of cat and nrf2 in tlr5alarvae compared to WT. The results of this study indicate that tlr5a knockout attenuates excessive inflammation and improves resistance against E. piscicida infection, likely by reducing bacterial adhesion and suppressing NF-κB-mediated pro-inflammatory pathways. This study highlights tlr5a as a potential immunomodulatory target to enhance disease resistance in teleost aquaculture.