Hemocyanin-mediated immunity in Pomacea canaliculata: Exploring a potential source of antimicrobial peptides

Hemocyanin respiratory pigments are recognized as key effectors of invertebrate innate immunity. Here, we further explore the immunological functions of hemocyanin from the freshwater snail Pomacea canaliculata (PcH), focusing on its potential as a source of cryptic antimicrobial peptides (AMPs) and its ability to mediate bacterial agglutination, combining in silico prediction, structural mapping, synthetic peptide assays, and functional immune tests. In silico analysis identified five PcH-derived peptide candidates with physicochemical features commonly associated with AMPs. Synthetic versions of these peptides inhibited the growth of the Gram-positive bacterium Staphylococcus pseudintermedius, and one peptide also exhibited activity against the Gram-negative bacterium Escherichia coli. These peptides exhibited bacteriostatic effects at low-millimolar concentrations with no detectable hemolytic activity against human erythrocytes. Structural mapping into PcH 3D cryo-EM structure indicated that all predicted AMPs are located in solvent-accessible regions, supporting their potential release by proteolytic processing. Additionally, native PcH exhibited dose-dependent agglutination of both Gram-positive and Gram-negative bacteria, consistent with the presence of exposed immunoglobulin-like domains, providing the first evidence in gastropods. Our findings support the view that PcH may be a multitasking immune effector and identify putative hemocyanin-derived AMPs in an Ampullariidae species, as well as evidence of hemocyanin-mediated microbial agglutinating activity in gastropods. Overall, this study highlights snail hemocyanin as a versatile component of molluscan innate immunity with potential as a source of bioactive peptides.