Functional Characteration Of Edwardsiella Tarda Twin-Arginine Translocation System

Edwardsiella tarda, a Gram-negative rod-shaped bacterium, infects a variety of host including human being. It is the aetiological agent of edwardsiellosis and responsible for systemic hemorrhagic septicemia in economic fish species such as turbot and flounder, leading to enormous economic losses in aquaculture worldwidely. Serection systems are competent for translocation of virulence fator and play a vital role in pathogenicity in bacteria. In addition, twin-arginine translocation （Tat） system is widely distributed in halophilic archaea and bacterial pathogens serving for extreme environments adaption and virulence factor secretion. It can translocate prefolded proteins into periplasm. Based on the completed work of E. tarda EIB202genomic sequencing, Tat system was detected on the genome of E. tarda EIB202. E. tarda Tat system gains more attention because of its adaptation to high-salt conditions （up to5%）. Meanwhile, there is no report on E. tarda Tat system so far.Tat system was composed by TatA, TatB, TatC, TatD, and TatE in E. tarda EIB202, and tatABCD was in one operon. In previous work, our lab has isolated or collected19E. tarda isolates and2E. ictaluri isolates from different area and host species. tatABCD was found to widely distribute in these tested Edwardsiella strains, while tatE was conserved in pathogenic strains. Meanwhile, the genetic relationship, polymorphism, and distribution of virulence genes of the21Edwardsiella strains were invesgated with biochemical characterization and molecular method. This will be helpful in the investigation of pathogenicity in Edwardsiella and the development of vaccines for edwardsiellosis.Tat system component null in-frame deletion strains AtatA, AtatB, AtatC, AtatD, AtatE, AtatAE, AtatABCD, and complement strains were constructed, and their roles in pleiotropically physiological fitness were analyzed in EIB202. Tat mutant displayed no difference with EIB202on growth, morphology, and biofilm formation. Except for AtatD and AtatE, five mutants of AtatA, AtatB, AtatC, AtatAAtatE, and AtatABCD displayed deficient on H2S production. AtatABCD was defective in a serious of functions, including the mobility on soft agar, the haemagglutination against rabbit erythrocyte, the autoaggregation in DMEM and LB culture, the hemolytic activity against sheep erythrocyte, the stress response such as high temperature, high-salt condition, SDS, ethanol. and antibiotics, the adherence to EPC and MDCK, the intracellular survival in J774a, and the marginal in vivo virulence attenuation in fish models.To further elucidate the underlining molecular mechanism for Tat system on physiological fitness, comparative proteomics analysis of whole cell lysated protein, periplasmic protein, and extracellular protein were implemented using two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry to identify proteins undergoing changes in expression levels when the Tat system was mutilated. In addition, a qualitative shotgun protein sequencing method was used for extracellular protein identification. The main abundance-different proteins were virulence-and stress-associated factors such as EvpF, FklB, FtsZ, ClpB, GrpE, PstS, ETAE₂022, DsbA, GlmU, and GapA. Interestingly, most of the identified proteins held non-classical Tat signal peptide and was associated with basal metabolism and biosynthesis of macromolecules. These would shed light on new mechanisms of the Tat system in E. tarda.The results of plate counting confirmed that the Tat mutant was high-salt-sensitive than EIB202, indicating that Tat mutant had merits as a novel salt-sensitive biological containment system for live attenuated vaccine in marine fish vaccinology. To test this, we deleted virulence-associated TTSS genes and cured endogenous plasmid pEIB202to construct strain WYM1in the context of Tat abrogation in E. tarda. The results indicated that WYM1was highly attenuated when injected intraperitoneally and elicited significant protection against challenge of wild-type E. tarda EIB202in turbot （Scophthalmus maximus） while being rapidly eliminated in seawater. Therefore, the environmental biosafety of live attenuated vaccine was improved.The Tat system translocated prefolded protein which would allow stabilization and prevent aggregation. Based on the live attenuated vaccine E. tarda WED, the Tat signal peptides mediated green fluorescent protein （GFP） secretion was analyzed with Western blotting. The signal peptides with optimized GFP translocation was further linked to gapA34, encoding a protective antigen glyceraldehyde-3-phosphate dehydrogenase （GapA） from fish pathogen Aeromonas hydrophila LSA34. Western blotting analysis illustrated that DmsA-GA, NapA-GA, and Sufl-GA displayed an optimal efficiency for translocation of GapA. The relative percent survival of the three constructive strains on turbot was assayed and DmsA-GA showed optimized relative protection rate （RPS） against E. tarda and A. hydrophila LSA34co-infection or single infection. Therefore, this study demonstrated a new approach for multivalent attenuated carrier live vaccine development.