title: Edwardsiella tarda-regulated proteins in Japanese flounder (Paralichthys olivaceus): Identification and evaluation of antibacterial potentials.
abstract: 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jing-jing
authors.initials: JJ
authors.lastname: Wang
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Collaborative Innovation Center of Deep Sea Biology, Zhejiang University, Hangzhou, China. Electronic address: lsun@qdio.ac.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D020609:Edwardsiella tarda / Q000302:isolation & purification* / Q000378:metabolism*; D004756:Enterobacteriaceae Infections / Q000378:metabolism / Q000382:microbiology / Q000662:veterinary*; D005393:Fish Diseases / Q000378:metabolism* / Q000382:microbiology / Q000517:prevention & control; D005432:Flounder; D006801:Humans; D008969:Molecular Sequence Data; D013154:Spleen / Q000378:metabolism; D016896:Treatment Outcome
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins
keywords: Antibacterial; Edwardsiella tarda; Paralichthys olivaceus; Proteomic