Felipe Pinto

Brazil

Deletion of natterin impairs NLRP3 inflammasome activation and host defense against Salmonella typhimurium in zebrafish

Felipe Justiniano Pinto1; Darlan Gusso1; Jefferson Thiago Gonçalves Bernardo1; Aline Ingrid Andrade-Barros1; Carlos DeOcesano-Pereira2; Monica Lopes-Ferreira1; Carla Lima1

1. Plataforma Zebrafish of the Laboratory of Applied Toxinology (CeTICs/FAPESP), Instituto Butantan, São Paulo, Brasil
2. Centre of Excellence in New Target Discovery (CENTD), Instituto Butantan, São Paulo, Brasil

Abstract

Background

BACKGROUND: Natterins are a family of proteins originally discovered in the venom of the fish Thalassophryne nattereri, but they are present in hundreds of other species, including zebrafish (Danio rerio). Although known for forming membrane pores and having an emerging role in immunity, their specific function in inflammasome activation remained unknown. The objective of this study was to demonstrate that zebrafish natterin (loc795232) is essential for NLRP3 inflammasome-mediated defense against bacterial stimulation.

Methods

METHODS: Using the CRISPR/Cas9 technique, we deleted the naterin gene in zebrafish embryos. These embryos were subsequently challenged with the bacterium Salmonella enterica serovar typhimurium (ST). We obtained responses through analyses of mortality, phenotypes, gene expression, immunohistochemistry, and enzymatic activity.

Results

RESULTS: Our results demonstrated that ST infection in zebrafish triggers sublethal phenotypes, such as depigmentation and hyperactivity, linked to immune dysregulation. Crucially, activation of the NLRP3 inflammation protects animals against mortality caused by ST; conversely, its inhibition increased lethality by blocking caspase activation and IL-1β release. The central discovery was that natterin is a critical regulator of this response. Knockout embryos (lacking the natterin gene) showed reduced expression of the nlrp3 and asc genes, impaired caspase (Caspy) activation, and an absence of mature IL-1β. Consequently, these embryos exhibited significantly higher mortality upon infection.

Conclusions

CONCLUSIONS: Our findings suggest that natterin plays a role in NF-κB-mediated priming and functional assembly of the inflammasome. Natterin appears to be a key modulator of innate immunity in teleosts, establishing a link between pore-forming proteins and IL-1β-mediated defense.