Characterization of a double WAP domain-containing protein from the red swamp crayfish Procambarus clarkii

Authors: ZHANG, HONG-WEI - University Of Missouri; MAN, XIN - Henan Normal University; WANG, YUE - Henan Normal University; SONG, QI-SHENG - University Of Missouri; Stanley, David; HUI, KAI-MIN - Henan Normal University; ZHANG, XIAO-WEN - Henan Normal University

Submitted to: Fish and Shellfish Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary: The red swamp crayfish is a freshwater animal originally found in northern Mexico and the southern United States. It is now a farm-reared food animal in North America and Asia. In China this crayfish is grown in combined rice-crayfish cropping systems. In some areas, such as the Great Lakes region it is an invasive species with moderate environmental impact. This problem with crayfish farming is they are susceptible to pathogens, including bacteria, fungi and viruses. These microorganisms seriously challenge crustacean agriculture. It follows that research into crayfish immunology is necessary. We are working to meet this global-scale need with discovery and analysis of new anti-microbial proteins. In this paper we report the discovery of an antimicrobial protein we discovered. Our analysis of the protein shows is it present in several tissues and infection with bacteria and viruses leads to increased concentrations of the protein. We also show that this protein kills bacteria. This is a meaningful step forward in crayfish immunology and our information will be valuable to other scientists conducting research into crayfish immunology. Ultimately, this new knowledge will benefit consumers of farm-reared crayfish at the global level.

Technical Abstract: Crustaceans express multiple whey acidic protein (WAP) domain containing proteins which are components of host immunity. In the present study, a new double WAP domain containing protein was identified from red swamp crayfish Procambarus clarkii, designated Pc-DWD. The ORF is 387 bp, encoding 128 amino acids consisting of signal peptide of 18 residues, and two tandem WAP domains of 38 and 44 residues. Multiple alignment indicates the presence of conserved motifs in both WAP domains, and phylogenetic analysis shows that Pc-DWD is a new member of the type-IV crustin family. Pc-DWD transcripts were found most abundantly in hemocytes, gills, intestine and heart, and induced by Vibrio anguillarum, Staphylococcus aureus and white spot syndrome virus challenge. RNAi knockdown of Pc-DWD expression led to increased expression of white spot syndrome virus genes and increased crayfish mortality after virus infection. Recombinant Pc-DWD exhibited strong protease inhibitory activity towards commercial subtilicin A and protease K. Pc-DWD inhibited the crude proteases from V. anguillarum and S. aureus cultures and from the crayfish tissue extracts. We infer that Pc-DWD acts in crayfish bacterial and viral immunity.