BNIP3, targeted by miR-148 and miR-152, upregulates autophagy to inhibit Aeromonas hydrophila infection in the giant freshwater prawn Macrobrachium rosenbergii

BNIP3 (Bcl-2/adenovirus E1B 19 kDa interacting protein 3) is a pro-apoptotic member of the Bcl-2 superfamily, that is implicated in autophagy during immune responses. To date, the mechanisms underlying BNIP3-mediated regulation of autophagy in crustaceans under pathogenic challenge remain poorly characterized. In this study, we demonstrated that BNIP3 in the giant freshwater prawn Macrobrachium rosenbergii (MrBNIP3), which is critical for immune defense, is essential for inducing autophagy during in vivo bacterial infection. The open reading frame of MrBNIP3 consists of 600 nucleotides that encode a 199-amino acid peptide containing multiple phosphorylation sites, a transmembrane domain, and a conserved BH3 motif. Phylogenetic analysis revealed high sequence similarity between MrBNIP3 and invertebrate BNIP3 orthologs as well as clustering within the crustacean clade. MrBNIP3 transcripts were ubiquitously expressed across all examined tissues, with maximal expression in muscle. Notably, MrBNIP3 expression was significantly upregulated in the gill and hepatopancreas following stimulation with the pathogenic bacterium Aeromonas hydrophila or lipopolysaccharide. RNA interference-mediated knockdown of MrBNIP3 suppressed autophagy activation and enhanced A. hydrophila invasion in M. rosenbergii. Furthermore, infection by A. hydrophila significantly downregulated microRNAs-148 and -152. Dual-luciferase reporter assays confirmed that these microRNAs co-targeted the 3′-untranslated region of MrBNIP3 to repress its expression. Collectively, these findings identify MrBNIP3 as a positive regulator of innate immunity in M. rosenbergii that is modulated through microRNA-dependent mechanisms.