Identification and Characterization of a Cathepsin B Protease in the Marine Fish Parasite Cryptocaryon irritans

Cryptocaryon irritans, an obligate ectoparasite, causes cryptocaryoniasis and heavy economic losses to marine aquaculture. Cathepsin B (CatB) is highly expressed in C. irritans theronts and hypothesized to participate in parasite host invasion, yet its biochemical properties and pathogenic roles remain poorly defined. In this study, we performed bioinformatic analysis of the CiCatB gene. Following codon optimization, CiCatB was heterologously expressed in Pichia pastoris, and the biologically active recombinant CiCatB protein was purified. Subcellular localization analysis revealed that CiCatB predominantly localized in the cytoplasm of theronts, with partial signals detected at the cell membrane and in the extracellular space. Enzymatic assays showed that yCiCatB hydrolyzed gelatin and type I collagen from large yellow croaker, with optimum activity at pH 5.0 and stable performance from 28 to 37 °C. Mg2+ increased enzyme activity, while Cu2+ almost completely abolished catalytic activity. Both E-64 and anti-CiCatB serum inhibited enzyme activity. Functional blocking using inhibitor or antiserum markedly reduced parasite recovery following challenge. Our results suggest that CiCatB may contribute to host invasion by degrading host structural proteins, advancing our understanding of C. irritans pathogenicity. CiCatB represents a potential target for exploring control strategies against cryptocaryoniasis.