| Edwardsiella tarda can infect farmed fish and develop edwardsiellosis, anenterohaemorrhagic septicaemic disease which result in huge economic losses inaquaculture. Vaccine immunization is an effective approach to prevent E. tardainfection. Attenuated vaccine could stimulate systemic immune response in hostadministrated by the route of immersion or oral, which is use-easy and efficacy in fishaquaculture. Attenuated vaccine is also be used as a vaccine carrier for thedevelopment of the polyvalent vaccine. In this research, I constructed11E. tardamutants by in frame deletion method, and tested their virulence and immuneprotection in fish. For development of attenuated E. tarda as vaccine carrier, weidentified the secretion signal region of EseG, an effector of E. tarda type threesecretion system (T3SS).1Characterization and immune protection of E. tarda LSE40ΔaroA mutantaroA gene encodes5-enolpyruvylshikimate-3-phosphate synthase, which isessential in aromatic amino acid synthesis. We constructed the aroA mutant of E.tarda LSE40and characterized its several phenotypes. Results showed that cellmorphology of the LSE40ΔaroA mutant was elongated and its cell division wasdelayed. This mutant showed lower growth rate in DMEM, less biofilm formation andsmaller swarming diameter. The mutant failed to aggregate as well as to secret threeT3SS translocator proteins (EseB, EseC and EseD) when cultured in DMEM medium.The virulence of LSE40ΔaroA was attenuated in turbot (Scophthalmus maximus) fishaffected by intramuscular injection (i.m.) or immersion. Growth of the mutant wasalso attenuated in turbot. The vaccinated fish administrated by i.m. or immersionshowed high agglutination titres against E. tarda in sera, however, no immuneprotection was observed for both vaccinated groups of fish.2Construction and virulence determination of E. tarda gene deletion mutantsEleven E. tarda LSE40gene deletion mutant were constructed by in framedeletion, including two single-gene-knockout mutants: LSE40ΔpurA and LSE40Δasd, seven double-genes-knockout mutants: LSE40ΔaroAΔesrB, LSE40ΔpurAΔesrB,LSE40ΔpurAΔaroA, LSE40ΔaroAΔesaC, LSE40ΔaroAΔrpoS, LSE40ΔaroAΔevpHand LSE40ΔesrBΔevpH, and two triple-genes-knockout mutants:LSE40ΔaroAΔesrBΔevpH and LSE40ΔaroAΔesaCΔevpH. Gene aroA encodes5-enolpyruvylshikimate-3-phosphate synthase, purA, adenylosuccinate synthase, asd,aspartate-semialdehyde dehydrogenase, rpoS, RNA polymerase sigma factor S, evpH,T6SS apparatus, esrB, T3SS regulator and esaC, T3SS apparatus. Blue gourami(Trichogaster trichopterus) were used to determine the50%lethal dose (LD50) ofthese mutants. Results showed that, these mutants have LD50which was444-1000times higher than the wild type E. tarda LSE40, indicating that all mutants wereattenuated.3Vaccination and immune protection of E. tarda mutantsFirstly, the immune protection of11mutants was evaluated in blue gourami bythe i.m. injection route. Results showed that two mutants, LSE40ΔaroAΔesaCΔevpHand LSE40ΔaroAΔesrBΔevpH did not showed detectable protection for fish; theremaining9mutants could protect blue gourami against wild type E. tarda infection,showing the relative percent of survival (RPS) ranging from21.7%to82.6%. Then,four mutants, LSE40ΔaroA, LSE40ΔaroAΔesrB, LSE40ΔaroAΔesaC, andLSE40ΔaroAΔevpH were evaluated for their immune protection in Japanese flounder(Paralichthys olivaceus) vaccinated by immersion route. Results showed that thespecific antibodies titres against E. tarda could be detected in the sera of vaccinatedfish. The RPS of the four mutants were0,45%,25%and20%against i.m. injectionchallenge of wild-type strain, and0,100%,66.7%and66.7%against immersionchallenge. The results showed that the LSE40ΔaroAΔesrB, LSE40ΔaroAΔesaC andLSE40ΔaroAΔevpH can provide good protection in flounder against wild-type E.tarda LSE40infection. Finally, the immune protection of LSE40ΔaroAΔesrB wasevaluated in turbot vaccinated by the route of immersion or i.m injection or oral.Results showed that immune protection could not be observed in the immersion andoral-vaccinated fish when challenged with wild-type E. tarda. The immune protectioncould be observed in the injection-vaccinated fish with doses of108and107cfu/ml,with RPS in20%and10%, respectively.4Identification the secretion signal of E. tarda T3SS effectorThe attenuated bacterial strains can be used as vaccine vectors for developmentof polyvalent vaccines. T3SS is a potential protein presenting system by delivering the foreign antigens into the host cell. In this study, the secretion signal peptide of T3SSeffector EseG and translocator EseC were predicted by bioinformatics analysis. Thepredicted regions for signal peptide were fused with reporter protein β-lactamase. Thefusion proteins were expressed in E. tarda and their secretion were detected bywesternblot. Result slowed that the N-terminal of1-88amino acid of EseG containedthe secretion signal peptide which could guide the secretion of fusion protein. Next,we will evaluate the potential of E. tarda T3SS as a foreign antigens delivery systemby the EseG signal peptide.
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