Edwardsiella tarda-regulated proteins in Japanese flounder (Paralichthys olivaceus): Identification and evaluation of antibacterial potentials

• Two-DE identified 20 spleen flounder proteins regulated by E. tarda infection.
• Cam, CatL, Crt, FerM, and NKEF were among the proteins upregulated by E. tarda.
• Overexpression of Cam, CatL, Crt, FerM, and NKEF inhibited E. tarda infection.
• Knockdown of CatL, Crt, FerM, and NKEF promoted E. tarda infection.
• Cam, CatL, Crt, FerM, and NKEF likely play a role in host defense against E. tarda.

Edwardsiella tarda is a severe aquaculture pathogen that infects a wide range of fish including Japanese flounder (Paralichthys olivaceus). In this study, proteomic analysis was conducted to examine flounder spleen proteins altered in expression during E. tarda infection. Twenty differentially expressed proteins were identified, which belonged to five functional categories. Five upregulated proteins, i.e. calmodulin (Cam), cathepsin L (CatL), calreticulin (Crt), ferritin middle subunit (FerM), and natural killer enhancing factor (NKEF), were evaluated for antibacterial potential. For this purpose, Cam, CatL, Crt, FerM, and NKEF were each overexpressed in flounder, and the ensuing effect on E. tarda infection was assessed. The results showed that overexpression of these proteins, in particular CatL, Crt, FerM, and NKEF, significantly inhibited bacterial dissemination in and colonization of fish tissues. To further examine their effects on E. tarda infection, CatL, Crt, FerM, and NKEF were knocked down in vivo. Subsequent analysis showed that knockdown of each of these genes significantly enhanced E. tarda invasion in flounder. These results indicate that E. tarda infection induced expressional changes in the proteins of diverse functions in flounder, and that some of these proteins function as immune defense factors that are required for effective clearance of invading E. tarda.Biological significanceThis study identified 20 flounder spleen proteins regulated in expression by E. tarda infection. Four of the upregulated proteins enhanced flounder resistance against E. tarda when being overexpressed and weakened flounder resistance against E. tarda when being knocked down. These results (i) provide the first global protein profile of flounder in response to E. tarda infection, (ii) identified flounder proteins that contribute significantly to the immune defense against bacterial pathogen, and (iii) demonstrate for the first time that calcium binding proteins, lysosomal protease, and ferritin of teleost are required for optimal antibacterial immunity.

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