The Eriocheir sinensis calcium/calmodulin-dependent protein kinase II activates apoptosis to resist Spiroplasma eriocheiris infection

Abstract

Calcium/calmodulin-dependent protein kinase II is a downstream mediator of calcium signalling and participates in the regulation of various cellular physiological functions. In previous studies, the expression of Eriocheir sinensis CaMKII (EsCaMKII) was significantly decreased in the thoracic ganglion after Spiroplasma eriocheiris infection, as shown using TMT-based quantitative proteomic analysis; however, the specific functions of EsCaMKII are still unclear. In this study, the full-length cDNA of EsCaMKII was 3314 bp long, consisting of a 1605 bp open reading frame encoding a protein of 535 amino acids, including a 258 aa serine/threonine protein kinase catalytic domain (EsCaMKII-CD). EsCaMKII is highly transcribed in haemocytes, nerves (thoracic ganglion), gills, and muscles, but lowly transcribed in the hepatopancreas, heart, and intestines. The transcription levels of EsCaMKII were altered in E. sinensis haemocytes after S. eriocheiris infection. After the over-expression of EsCaMKII-CD in RAW264.7 cells, the apoptosis rate of RAW264.7 cells was significantly increased. After the over-expression of EsCaMKII-CD, the morphology of RAW264.7 cells became worse after being infected with S. eriocheiris. Meanwhile, the copy number of S. eriocheiris in RAW264.7 cells was significantly decreased. From 48 h to 96 h after EsCaMKII RNA interference, the transcription levels of EsCaMKII decreased significantly. The transcription of apoptosis genes and cell apoptosis were also inhibited in haemocytes after EsCaMKII RNAi. The knockdown of EsCaMKII by RNAi resulted in significant increases in the copy number of S. eriocheiris and in the mortality of crabs during S. eriocheiris infection. These results indicate that EsCaMKII could promote the apoptosis of E. sinensis and enhance its ability to resist S. eriocheiris infection.