| Edwardsiella tarda is a commonly found pathogen in aquaculture, which can infect manyimportant economic fish. The outbreak of edwardsiellosis leads to mass mortality in variouspopulations and age groups of fish, resulting in significant losses to the aquaculture industry.Thus far, research on the mechanism of E. tarda has already involved in many aspects,including flagellin, invasion, lipopolysaccharides, hemolysin, glycosyltransferases, type III/VI secretion system, siderophores, QseB-QseC two component system and so on. However,the exact mechanism is still not clear. Currently, in aquaculcuture, antibiotics are still themain way to treat edwardsiellosis. The largely use of antibiotics may lead to the rising ofresistant strains, making it more difficult in fighting with edwardsiellosis. Therefore, athorough understanding of the mechanism of Edw.tarda and the development of effectivevaccine is very urgent.Capsule is an important virulence factor of bacteria, which can promote adherence ofbacteria to host cells，in the meantime, it can also protect bacteria from the immune systemkilling. An interesting phenomenon was found in our previous work; under normal culturecondition, there was no capsule structure in E. tarda, but the capsule structure was observedafter infected the macrophages of flounder. Therefore, we assumed that there might be acorrelation between pathogenic and capsule. In this study, three genes involved in thesynthesis of capsule were investigated, including rcsB (the response regulator of Rcsphosphorelay), wcaJ (a transferase in the synthesis of colanic acid) and cpsB (aMannose-1-phosphate guanyltransferase in the synthesis of colanic acid). Three in-framedeletion mutants were constructed through double-crossover allelic exchange. Briefly, theupstream and downstream fragments of the three deletion genes were amplified by PCR,respectively; Overlap PCR was conducted to fuse the upstream and downstream fragments; The fused fragments were then cloned to the suicide vector pRE118; The suicide vector whichcarry the fusion fragments were transformed into E. coli SY327and then introduced into E.coli S17-1for mating into E. tarda EIB202by conjugation. According to Holliday model,in-frame deletion mutants were constructed through double-crossover allelic exchange. Bytransformation of a low-copy plasmid pACYC184K carrying the intact deletion gene,complementary strain was constructed. In the following experiments, some virulence-relatedphenotypes were tested, including biofilm formation, lipopolysaccharides (LPS),autoagglutination, adherence and internalization to Epithelioma papulosum cyprini (EPC)cells and the lethality to zebrafish embryos. These results are as follows:(1) In this study, an in-frame deletion mutant of rcsB in E. tarda EIB202was constructedusing the suicide vevtor pRE118, then its complementary strain was constructed byelectroporating a low copy plasmid pACYC184K into the deletion mutant. Severalvirulence-related phenotypes were tested. Compared with the wild-type strain EIB202,biofilm formation decreased significantly in ΔrcsB, while ΔrcsB (pACYC184K-rcsB)recovered the phenotype to some extent. In addition, the ability of autoagglutination, thepercentage of adherence and internalization to EPC cells and the lethality to zebrafishembryos significantly increased in ΔrcsB. All these phenomena displayed by the mutantΔrcsB had a certain degree of recovery, but not completely, in strain ΔrcsB(pACYC184K-rcsB), which indicated that the deletion gene of rcsB accounts for thesechanges of ΔrcsB. The present results indicated that rcsB was involved in the growth ofbacteria, biofilm formation, the autoagglutination ability, invasion to EPC cells and zebrafishembryos. In addition, the results also indicated that the pathogenic process of bacteria iscomplicated, and an evaluation of the virulence of bacteria cannot simply rely on biofilmformation, the growth pattern or other unilateral considerations.(2) By overlap PCR and double-crossover allelic exchange, the in-frame deletion mutantΔwcaJ was constructed. The complementary strain ΔwcaJ (pACYC184K-wcaJ) was constructed by transformation a low-copy plasmid pACYC184K carrying the intact wcaJ.Several virulence-related phenotypes were tested. It was found that the deletion of wcaJpromoted the bacterial growth. However, the deletion mutant showed reduced biofilmformation, LPS production, adherence and internalization to EPC cells and pathogenicity tozebrafish embryos. All the phenotypes displayed by the deletion mutant were recovered in thecomplementary strain, which indicated that the changes of these phenotypes resulted from thedeletion of wcaJ. These results revealed that wcaJ was involved in the growth of bacteria, theLPS production, biofilm formation and virulence to EPC cells and zebrafish embryos. Thisresearch also demonstrated that the relationship between the growth of bacteria and virulencemight not necessarily a positive correlation.(3) Similarly, an in-frame deletion mutant ΔcpsB and a complementary strain ΔcpsB(pACYC184K-cpsB) were constructed. Several virulence-related phenotypes were tested.Compared with the wild-type strain E. tarda EIB202, the abilities of biofilm formation, LPSproduction, adherence to EPC cells and the lethality to zebrafish embryos were weakened inthe deletion mutant ΔcpsB, but the growth of bacteria showed no difference compared withthe wild-type strain. These phonotypes had a certain degree of recovery in complementarystrain ΔcpsB (pACYC184K-cpsB), which indicated that the changes of these phenotypesresulted from the deletion of cpsB. These results also indicated that cpsB was involved in theLPS production, biofilm formation and virulence to EPC cells and zebrafish embryos.This is the first report that capsule polysaccharide has a virulence-related function in E.tarda. This research provides useful information for further studies on pathogenesis of E.tarda. |
PDF Full Text Request