Abstract

The NLRP3 inflammasome is essential for innate immunity against pathogens, yet its function in amphibians remains poorly understood. Here, we identified and cloned two novel isoforms of NLRP3 from the American bullfrog ( Aquarana catesbeiana ), designated Ac NLRP3 X1 and Ac NLRP3 X2, with open reading frames of 2397 bp (798 aa) and 2874 bp (957 aa), respectively. Both isoforms contain conserved NACHT and LRR domains, lack a typical PYD domain, and share high structural similarity to other amphibians' NLRP3 homologs. Tissue distribution analysis revealed that both isoforms are broadly expressed in immune-related organs and are significantly upregulated upon infection with Citrobacter freundii , Elizabethkingia miricola , Aeromonas hydrophila , and Edwardsiella tarda . In vivo knockdown of either isoform significantly reduced the transcript levels of downstream inflammasome-related genes, including Ac ASC, Ac Caspase1, Ac IL-1β, and Ac IL-18. Collectively, these findings demonstrate that both Ac NLRP3 isoforms contribute to antibacterial immunity by regulating inflammasome signaling, providing insight into amphibian innate immunity and expanding understanding of NLRP3 function across vertebrates.