| Edwardsiella tarda is one of the important pathogens in aquaculture, and has caused serious loss of marine cultured fish such as turbot (Scophthalmus maximus)and Japanese flounder (Paralychthys olivaceus) in Shandong Province, China. A fish pathogen Edwardsiella tarda LTB-4 has been reported to produce various indole alkaloids, indole was the most abundant among these indole alkaloids. Currently, indole has been proved to be a new quorum sensing signal molecule, and control diverse aspects of bacterial physiology, such as amino acid metabolism, spore formation, plasmid stability, drug resistance, biofilm formation, virulence and acid resistance. In previous studies, indole was found to not only regulate motility, lipopolysaccharide production and drug resistance of Edw. tarda, but also involve in the pathogenicity to zebrafish. Type Ⅲ secretion system is an important virulence secretion system of Edw. tarda,the signal transduction pathway of indole in type Ⅲ secretion system of Edw. tarda has not yet been reported yet.In this study, Δ, ΔtnaAΔAesrB, ΔcpxR, Δand ΔtnaAΔcpxR mutants were constructed using the method of overlap-PCR. Combined with real-time quantitative PCR (Real time PCR), the effect of indole on the expression of type Ⅲ secretion system related genes, baeR and cpxR genes in two-component regulatory systems was explored. We further investigated the interaction between indole production and the expression of rpos gene, the effect of indole on acid resistance in Edw. tarda. Finally, the virulence of these mutants to zebrafish was also explored by in vivo pathogenic assays.As a result, type Ⅲ secretion system related genes (esaC, escA, eseC, eseE, eseG, esrA, esrB and esrC) were significantly induced by tryptophanase encoding gene (tnaA), except several genes(escB, escC, eseB and eseD). Notably, the expression ofesrB in LTB-4 showed 32-fold higher than AtnaA mutant. Indole signalling was transmitted via regulator gene esrB to regulate the expression of type III secretion system related genes,but esrB is not the only signal transduction pathway of indole. The expression of type Ⅲ secretion system related genes were induced by indole in a concentration-dependent manner,30μM indole (the maximum physiological concentration of indole producingby Edw. tarda are 35.5μM) induced the maximal expression level of type Ⅲ secretion system related genes. In addition, PhoP-PhoQ two-component system which is closely associated with type III secretion system was not regulated by indole.Also, indole positively regulated the expression of regulator genes cpxR and baeR in Edw. tarda, but only regulator gene cpxR was affected by indole to further induce the expression of type Ⅲ secretion system related genes. But indole could still regulate the expression of type Ⅲ secretion system related genes without regulator gene cpxR. Since the expression of genes (esrA、esrB and esrC) in ΔcpxR mutant was significantly lower than AtnaA mutant, indole was speculated as one of the important signaling molecules of CpxAR two-component regulatory system. Except AbaeR mutant, attenuated virulence of the other mutants (AesrB, AcpxR, ΔtnaAAcpxR and AtnaAAesrB) by significantly increasing the survival rate of zebrafish indicated that indole induced type III secretion system via CpxAR two-component regulatory system and further regulated the virulence of Edw. tarda.In this study, low dose of Carbenicillin was found to promote Edw. tarda to produce more indole and significantly inhibit the gene expression of rpos. And the expression level of rpos in ΔtnaA mutant was highest in response to OμM indole, which indicated that indole inhibited the gene expression of rpos. However, the expression level of tnaA in Arpos mutant was significantly lower than LTB-4(P<0.01) and Δrpos mutant decreased the indole production.Compared to LTB-4, the expression of genes gadBl and gadB2 (glutamate decarboxylase encoding gene) in AtnaA mutant was significantly induced (P<0.01). And the survival rate of LTB-4 in pH 2.5 was much lower than that of ΔtnaA mutant. The result indicated that indole decreased the acid resistance of Edw. tarda.The study of signal transduction pathways of indole in type III secretion system in Edw. tarda will help us to understand the role of indole in pathogenic mechanism in Edw. tarda, and develop a new environmentally friendly anti-infective strategy in aquaculture. |
PDF Full Text Request