Molecular identification of a calmodulin gene from Cryptocaryon irritans

Cryptocaryon irritans is a parasitic ciliate causing ‘white spot disease’ on marine teleosts that often results in huge financial losses to mariculture. So far, there is no safe and effective method to control the parasite infection. Calmodulin (CaM), as a sensor for Ca²⁺ signaling, regulates cell movement, cell division and invasion of some protozoan parasites to their hosts. To understand the role of calmodulin in the growth and development of C. irritans, the calmodulin gene (cicam) was cloned from the C. irritans trophozoite cDNA library, and the open reading frame (ORF) was synthesized after codon optimization. The recombinant plasmid pGEX-4T-1/cicam was constructed, then transformed into Escherichia coli and induced its recombinant expression with ZYM-5052 self-inducing medium. The recombinant protein rCiCaM was purified by glutathione agarose gel and thrombin, and obtain polyclonal antibodies by immunizing with the mouse BALB/c strain. The expression of cicam and its encoded protein CiCaM in each stage of the worm was examined by reverse transcription PCR and immunoblotting assay, respectively. The localization of CiCaM in the larvae was examined by indirect fluorescent antibody assay. The activity of recombinant protein rCiCaM binding recombinant actin-depolymerizing factor was initially investigated by blot overlay assay. The results showed that the open reading frame (ORF) of CiCaM was 450 bp, which encoded a polypeptide of 16.9 ku, consisting of 149 amino acids; the molecular masses of GST-rCiCaM and rCiCaM were 43 ku and 16.9 ku respectively, which corresponded with the predicted ones; the CiCaM gene expressed in all developmental stages of C. irritans, and the molecular mass of the native CiCaM corresponded to the predicted value; the CiCaM distributed all over the cytosol of C. irritans theronts, especially abundant around the four macro nuclei and the peripheral area of cytostome; rCiCaM could interact with rCiADF2 in a Ca2+-dependent way. This study enriched the knowledge of molecular biology of the pathogen C. irritans, which would provide a reference for the prevention and control of cryptocaryonosis.