Differential Antibacterial and Host-Associated Effects of Full-Length Procambarus clarkii PcALF2 and Its LBD during Aeromonas hydrophila Challenge

Antilipopolysaccharide factors (ALFs) are key crustacean antibacterial effectors, but differences between full-length ALFs and isolated lipopolysaccharide-binding domains (LBDs) remain unclear. Here, we compared recombinant PcALF2 (rPcALF2) with synthetic LBD2 against Aeromonas hydrophila. Both exhibited antibacterial activity and membrane disruption, but rPcALF2 consistently exhibited stronger inhibition. Competition assays with lipopolysaccharide (LPS) and cardiolipin indicated shared LPS-targeting activity, whereas rPcALF2 displayed stronger interactions with cardiolipin-associated membrane targets. Transcriptomic analysis revealed distinct response patterns: LBD2 induced stress-adaptation pathways, whereas rPcALF2 was associated with suppression of ribosome function, oxidative phosphorylation, and pyrimidine metabolism. In Procambarus clarkii, PcALF2 silencing impaired bacterial clearance and exacerbated tissue damage, whereas exogenous rPcALF2 conferred greater protection than LBD2. Intestinal 16S rRNA analysis showed that rPcALF2 was associated with lower Proteobacteria and higher Bacteroidota/Firmicutes than LBD2. These findings indicate that structural elements beyond the isolated LBD contribute to the stronger antibacterial and host-protective effects of full-length PcALF2.