Investigation Of The Edwardsiella Tarda T3SS Translocon Proteins And Chaperone

Edwardsiella tarda is an important gram-negative pathogen affecting both animals and humans. Edwardsiellosis, caused by E. tarda, has been found in many commercially important cultured fish, and has led to extensive losses in both freshwater and marine aquaculture annually. Till now chemical methods are commonly used for prevention against edwardsiellosis, while related studies of vaccines are still on the road. This pathogen harbors a type III secretion system (T3SS) essential for bacterial pathogenesis. Although the structure of T3SS gene cluster together with the function of some genes in T3SS have been primarily studied, its mechanism hasn't been illuminated yet. In this experiment, we focus our study on the identification and investigation of the chaperone for the E. tarda T3SS translocon protein EseC, and also carry out primary research for translocon proteins and their immunoprophylactic potential to fish, trying to learn more about the mechanism of T3SS in the pathogenesis of E. tarda and in the development of vaccine.1. Identification and investigation of the chaperone for the E. tarda T3SS translocon protein EseCAs previously reported, EseB, EseC, and EseD are the components of the T3SS translocon, and were shown to form this complex after secretion. Chaperones are important for the stabilization and secretion of T3SS translocon proteins. EscC functions as a T3SS chaperone for EseB and EseD, whereas the chaperone for EseC hasn't been reported. In this experiment, we took EseC as the research object, and identified the chaperone of EseC.Bioinformatics assay showed that escA gene is located proximately to eseC. EscA is a small (molecular mass 17.5 kDa) an acidic (pI 4.79) protein, having a large helix configuration, and is homologous to the identified chaperones, all of which fulfill the common characteristics of a T3SS chaperone. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC; meanwhile the accumulation of EseC in the cytoplasm was also affected. While escA mutant was complemented by escA gene, the secretion and accumulation of EseC were resumed to the wild type secretion phenotype. As we used chloramphenicol to block the production of new proteins, we observed that EseC protein gradually degraded without the existence of EscA, showing EscA is important for the stabilization of EseC. Co-purification studies demonstrated a specific interaction between EscA and EseC in vitro, while co-immunoprecipitation studies proved that EscA could bind to EseC in vivo. Hereto we conclude that EscA functions as the chaperone for EseC.Having confirmed the chaperone role of EscA for EseC, we further studied the affection of EscA to the expression of EseC, and the binding region of these two proteins. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC by construction and measuration of an EseC-LacZ fusion protein in E. tarda. By constructing and Western blot analyzing in vitro co-expression systems containing in-frame mutations of partial sequence of EscA and EseC, we found that residues 31–137 of EseC are required for EseC-EscA interaction, while we couldn't find a similar region in EscA particularly used for binding EseC. We constructed the ?eseC31-137 mutant and found that absence of EseC residues 31–137 reduced the secretion and accumulation of EseC at a similar level as escA deletion in E. tarda, attesting that 31-137 regions are the binding domain of EseC to EscA. Finally, artificial infection experiments showed that EscA and its interaction with EseC contribute to the virulence of E. tarda.2. Study of the E. tarda T3SS translocon proteinsAs previously reported, T3SS proteins could express when bacteria contact with their host, as well as bacteria are induced in vitro. We checked the expression of E. tarda T3SS translocon proteins in different temperatures and different pH values, in order to confirm the preferable induction condition. As the results, in 37°C, E. tarda grew fastest, but the expression of translocon proteins was lower; in 28°C, the expression of translocon proteins was highest; in 20°C, we couldn't detect their expression. Meanwhile alkaline and neutral environments are better that acidic culture for growing and T3SS expressing. We analyzed the cellular fractionation of three Ese proteins in wild type strain as well as translocator mutants, and deduced the possible mechanism of the comformation of translocon. Absence of single translocon component protein doesn't affect the expression of other two, while the secretion of translocon proteins could interact in some extent.We used the ?eseB, ?eseC and ?eseD mutants by measuring their growth conditions as well as their swimming motilities, autoaggregate abilities and hemolysis abilities, and found that in the deletion of translocon proteins, the growth of bacteria is slower than wild type, the abilities of motility, autoaggregate and hemolysis also reduced, demonstrating the important roles of the translocon proteins for the growth and function of E. tarda.To test the immunoprophylactic effect of translocon protein, the eseD gene was cloned, expressed in E. coli BL21 (DE3) strain, and recombinant EseD protein was purified by Ni-NTA resin. Scophthalmus maximus was immunized using the purified protein and developed antibodies with a climax titer of 1:5120 on the 7th week, showing that the protein is highly immunogenic in fish. Challenge experiment of E. tarda demonstrated that the protein is effective for preventing E. tarda infection, as the relative percent survival (RPS) of turtle fish was 62.5% when the concentration of E. tarda was 105cfu. The EseD protein shows promise as vaccine candidate in aquaculture and could probably be used for the protection of fish against edwardsiellosis.