# Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1/Gbp4 to license Caspy2-mediated antibacterial immunity

## TL;DR

Natterin helps zebrafish fight Salmonella by linking interferon and inflammasome pathways, which is crucial for survival.

## Contribution

Natterin is identified as a master regulator connecting IFN-I and inflammasome signaling in teleost immunity.

## Key findings

- Natterin deficiency abrogates IFN-I response and prevents GBP4 and Caspy2 activation.
- IFN-I neutralization in wild-type embryos mirrors the Natterin knockout phenotype.
- Natterin is essential for coordinating immune responses against Salmonella Typhimurium.

## Abstract

The Natterin protein family represents an evolutionarily conserved group of immune effectors in teleosts, yet its broader regulatory role in host defense remains poorly understood. Here, we demonstrate that Natterin functions as a master upstream regulator, orchestrating a critical immune network that integrates type I interferon (IFN-I) signaling with non-canonical inflammasome activation during Salmonella Typhimurium (ST) challenge. Using wild-type embryos treated with IFN-I neutralizing antibody followed by the use of natterin (loc795232) knockout (KO) embryos generated by CRISPR/Cas9 and integrated approaches—including RT-qPCR, Western blotting, immunohistochemistry, and behavioral assays—we found that its absence completely abrogates the ST-induced IFN-I response, including the ablation of the interferon regulatory factors irf3 and irf7 and the IFN-φ1 receptor crfb1. Consequently, Natterin deficiency prevented the expression of the LPS sensor GBP4 and the proteolytic maturation of the inflammatory caspases Caspy and Caspy2. This disruption abolished downstream gsdme-a/b expression, which may result in the non-formation of pores. The critical role of IFN-I signaling was independently confirmed by its neutralization in wild-type embryos, which abolished the protein-level localization of IL-1β and IFN-β and mirrored the KO phenotype. Functionally, this disruption led to a sixfold increase in mortality and exacerbated ST-induced pathogenesis. Our results establish Natterin not merely as an effector molecule but as a pivotal regulator that integrates IFN-I and inflammasome signaling, orchestrating a coordinated immune response essential for host survival. This work reveals a previously unrecognized level of regulation in teleost innate immunity with significant evolutionary parallels to mammalian defense mechanisms.

Proposed mechanism for Natterin-dependent inflammasome activation in zebrafish defense against Salmonella Typhimurium (ST). Natterin is essential for coordinating a protective immune response against ST by bridging type I interferon (IFN-I) signaling to inflammasome activation. It regulates the IRF3-7/crfb1 axis to drive the production and binding of IFNφ1, respectively. This IFN-I response is a critical prerequisite, as it enables the assembly of a Caspy-Caspy2/GBP4 inflammasome complex for cytosolic LPS sensing and subsequent maturation of IL-1β, gasdermin-E activation, pore formation and the release of IL-1β. GSDME pores also facilitate K+ efflux. The essential role of this pathway is underscored by the finding that neutralization of IFN-I phenocopies the immune impairment observed in natterin-knockout embryos, abolishing inflammasome activation and IL-1β–mediated immunity.