Characterization of cathepsin D from Eriocheir sinensis involved in Spiroplasma eriocheiris infection.

Cathepsin D (catD) belongs to a lysosomal aspartic protease superfamily. The full-length catD cDNA from the Chinese mitten crab Eriocheir sinensis (EscatD) was 2748 bp and contained a 1158-bp ORF encoding a protein of 385 amino acids, including a signal peptide and two N-glycosylation sites. Phylogenetic analysis showed that EscatD was clustered into a single group, together with other catD for crustaceans. Quantitative real-time PCR revealed that EscatD was expressed mainly in the eyes, hemocytes, intestine and nerve and was expressed weakly in heart, muscle and gills. After challenge with Spiroplasma eriocheiris, the expression of EscatD was significantly up-regulated from 1 d to 9 d. The copy number of S. eriocheiris in a silencing EscatD group was significantly higher than those in the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in an EscatD-dsRNA group. We further found that knockdown of EscatD by RNA interference resulted in a downward trend of expression levels of JNK, ERK, relish and p38 during the early stage, as well as a reduction in the expression of five antimicrobial peptides genes, namely, crusrin1, crustin2, ALF1, ALF2 and ALF3. The subcellular localization experiment suggested that recombinant EscatD was mainly located in the cytoplasm. The over-expression in Drosophila S2 cells indicated that EscatD could decrease the copy number of S. eriocheiris and increase cell viability. The above results demonstrated that EscatD plays an important immune role in E. sinensis to S. eriocheiris challenge.