Heterologous expression, enzymatic activity, and functional characterization of Cathepsin L from tomonts of...

Increased abundance and vitality of Cryptocaryon irritans lead to more severe pathogenicity in marine fish, and inhibition of C. irritans tomont hatching represents a promising therapeutic strategy for combating this parasite. Cathepsin L, a protease implicated in tomont development, was investigated to elucidate its regulatory role in tomont hatching. We cloned and characterized the full-length cDNA of C. irritans cathepsin L, heterologously expressed the protein in Escherichia coli, and quantified its enzymatic activity. CRISPR/Cas9 knockout mutants of C. irritans were generated using two single-guide RNAs (sgRNAs, C1 and C2), which induced frameshift mutations via a 2-bp deletion (C1) and a 4-bp deletion (C2). Compared with the control group, these mutations caused a 75.4% and 87.1% reduction in cathepsin L mRNA expression, 48.4% and 79.6% decrease in enzymatic activity, and a significant reduction in protein abundance. The recombinant cathepsin L exhibited maximal proteolytic activity toward the fluorogenic substrate Z-Phe-Arg-AMC at pH 6.0 and 40 °C, and its activity was significantly inhibited by Cu²⁺ and leupeptin. Phenotypic analysis showed that after 36 h of in vitro culture, tomonts in both knockout groups exhibited extensive developmental arrest. The hatching rates of the C1 and C2 groups decreased by 60% and 72%, respectively, which were significantly lower than the control group (p < 0.01). These results confirm that knockout of cathepsin L in C. irritans effectively blocks tomont development. Furthermore, our optimized electroporation protocol markedly improved the CRISPR/Cas9 editing efficiency in this hard-to-transfect parasite, providing a practical and reliable approach for functional gene studies in C. irritans.