A Study On The Immune Evasion Mechanism Of Edwardsiella Tarda

Edwardsiella tarda is a Gram-negative bacterial pathogen that can infect a wide range of freshwater and marine fish, and has become a significant limitation on the development of aquaculture. The complicated pathogenesis of E. tarda has led to a huge difficulty for the treatment of edwardsiellosis. Now, more and more researchers have been committing to the pathogenesis of E. tarda and the immune response mechanism of host. In the present study, using a series of in vitro and in vivo experiments, we investigated the pathogenesis that how E. tarda can have the ability to inhibit apoptosis and survive in host serum. In addition, we analyzed the functions of a C type lectin in Cynoglossus semilaevis in response to the pathogens infection.Firstly, we observed that E. tarda replicated robustly in the zebrafish cell line ZF4, and that E. tarda-infected cells exhibited no detectable signs of apoptosis. Global transcriptome analysis and quantitative real-time RT-PCR revealed that E. tarda infection generally significantly downregulated pro-apoptotic genes and upregulated anti-apoptotic genes. To investigate the role of apoptosis in E. tarda infection, two upregulated anti-apoptotic genes(Fech and Prx3) and two downregulated pro-apoptotic genes(Brms1a and Ivns1a) were overexpressed in zebrafish. Subsequent infection study showed that Fech and Prx3 overexpression significantly promoted E. tarda dissemination in and colonization of fish tissues, while Brms1 a and Ivns1 a overexpression significantly reduced E. tarda dissemination and colonization. Consistently, when Fech and Prx3 were knocked down in zebrafish, E. tarda infection was significantly inhibited, whereas Brms1 a and Ivns1 a knockdown significantly enhanced E. tarda infection. These results indicate for the first time that E. tarda prevents apoptosis in teleost as a strategy for intracellular survival.Secondly, in order to identify E. tarda proteins involved in serum resistance, we conducted proteomic analysis to examine the extracellular protein profiles of TX01, a pathogenic E. tarda isolate, in response to serum treatment. Five differentially expressed proteins were identified, one of which was a putative zinc protease(named Sip1). Western blot confirmed extracellular production of Sip1 by E. tarda. Sequence analysis revealed that Sip1 possesses a conserved zinc metalloprotease motif and shares low homology with the putative zinc proteases/aureolysin of several bacterial species. Purified recombinant Sip1(rSip1) exhibited zinc-dependent proteolytic activity that reached maximum at 40?C and pH 8. Compared to the wild type, the sip1 knockout mutant, TX?sip1, was dramatically reduced in the ability to cause mortality in the host(Japanese flounder) and to survive in host serum. These lost virulence capacities of TX?sip1 were restored by complementation with the sip1 gene. Further study showed that rSip1 enhanced the serum resistance of TX01 and TX?sip1, whereas antibody blocking of the Sip1 produced naturally by TX01 impaired serum resistance. Vaccination study showed that rSip1 as a subunit vaccine was able to induce effective protection in flounder against E. tarda challenge. Taken together, these results indicate that Sip1 is a novel zinc metalloprotease that is essential to serum resistance and host infection.Finally, many C type lectins(CTLs) have been identified in teleost, however, the in vivo function of fish CTLs are essentially unknown. In this study, we examined the function of a CTL(CsCTL1) from tongue sole(C. semilaevis). CsCTL1 possesses the conserved EPN motif required for mannose binding in mammals but unknown in function in fish. CsCTL1 is composed of 169 residues and shares low to moderate(31.2%-53.2%) sequence identities with the CTLs of other teleost species. Expression of CsCTL1 occurred predominately in liver and was upregulated during bacterial and viral infection. Recombinant CsCTL1(rCsCTL1), but not the mutant rCsCTL1 M bearing substitutions at EPN, interacted with and agglutinated a limited range of bacteria which ddid not include E. tarda. The agglutinating ability of rCsCTL1 was abolished in the absence of calcium or presence of mannose. Binding of rCsCTL1 to bacteria promoted phagocytosis and antimicrobial activity of head kidney monocytes. Fish administered with rCsCTL1 exhibited enhanced resistance against bacterial and viral infection. These results provide the first evidence that the EPN site is essential to a fish CTL and that, in addition to antibacterial properties, a fish CTL promotes the immune defense against viral infection as well. However, E. tarda was able to evade the antibacterial effect induced by CTL through not being bound by CTL.