Target genes directly regulated by Eha are required for Edwardsiella tarda survival within macrophages.

Eha is a virulence regulator in Edwardsiella tarda (E. tarda). The present study examined how Eha regulated its target genes to affect the bacterial survival within the cells. We constructed the reporter a pGEX-4T-ehaflag plasmid expressing Eha tagged at its C terminus with the flag epitope, and introduced the plasmid into an eha mutant ET13 strain, and obtained a Cehaflag strain. The expression and activity of an EhaFlag fusion protein restored the survival of the Cehaflag as the wild type in macrophages by Western blotting and intracellular survival experiments. We used a monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complexes using chromatic immunoprecipitation (ChIP). We then designed primers based on the differentially-expressed genes identified from RNA-sequencing, and identified ten Eha-interacting genes by qPCR. We amplified the promoter regions of the ten genes and the eha gene from ET13 strain by PCR, constructed pBD-PtargetlacZ and pBD-PehalacZ plasmids. The eha gene directly and positively regulated these target genes, and be negatively auto-regulated by Eha in E. tarda, as determined by comparing their ß-Galactosidase activities. These target genes were distributed in the categories involved in the bacterial growth, movement and resistance to H2O2 or acid. We further constructed a ETATCC_RS15225 mutant (△dcuA1), a ETATCC_ RS14855 mutant (△flgK) anda ETATCC_RS07650 mutant (ΔtnaA), and a partial complementary strains of △eha-tnaA and △eha-flgK and the complementary strains of CΔflgK and CΔtnaA. The ETATCC_RS15225 gene probably encoded a transporter protein DcuA1 at outer membrane with SDS-PAGE and RT-PCR. The ETATCC _RS14855 gene probably encoded FlgK protein and affected the bacterial motility. The ETATCC_RS07650 gene encoded Tryptophanase, which affected the bacterial survival within macrophages. With the assistance of these above strains, our results showed that the eha gene was able to regulate the ETATCC_RS15225 gene to express its outer membrane protein DcuA1, the ETATCC _RS14855 gene to control the flagellar motility and the ETATCC_RS07650 to affect the bacterial survival within macrophages. With the combination of other functions of above three genes, our results suggested that Eha directly regulates the target genes to affect E. tarda to survive within the cells.

Eha, a regulator of Edwardsiella tarda, required for resistance to oxidative stress in macrophages.

Edwardsiella tarda is distributed widely in a variety of hosts. Eha has recently been found to be its virulence regulator. In order to explore the mechanism of its regulation, we investigated the survival rates of wild type strain ET13, and its eha mutant and complemented strains in RAW264.7 macrophages under light microscopic observation as well as by counting bacterial CFUs on the plates. All of the different strains could live within the macrophages; however, the intracellular numbers of the wild type were significantly higher than the mutant when the incubation time extended 4 h or 6 h (P < 0.05). Furthermore, more ROS were produced by the mutant-infected cells, indicating that Eha may enhance ET13's capacity to detoxify ROS. In agreement with this, we found that the mutant exhibited more sensitivity by H2O2 disk inhibitory assay and less survival ability with H2O2 treatment. We further demonstrated that the bacterial antioxidant enzymes SodC and KatG were regulated by Eha with qRT-PCR and ß-galactosidase assay. Collectively, our data show Eha is required for E. tarda to resist the oxidative stress from the macrophages.

The role of regulator Eha in Edwardsiella tarda pathogenesis and virulence gene transcription.

Edwardsiella tarda is a pathogen with a broad host range that infects both animals and humans. Eha is a new transcriptional regulator identified in ET13, which is involved in the bacterial hemolytic activity. This study explored the effect of the Eha in the pathogenesis of E. tarda and the transcriptional regulation of the bacterial virulence genes (eseC, fliC, pagC and fimA). Our results found that the virulence of the eha mutant was 2.5-fold less than the one of its wild ET13 by LD50 in a murine model of i.p. infection, and the bacterial loads of the mutant displayed a different profile from the one of the wild strain. Most significantly, the mice infected with the mutant have greatly reduced acute inflammation in the liver, spleen and kidney compared to the ones infected with the wild. We further demonstrated that eseC, fliC and pagC were regulated directly by the Eha with qRT-PCR and ß-Galactosidase assay, but fimA wasn't done. The promoter regions of the genes modulated and the cly gene reported before had been found to contain a common conserved motif by using software. In addition, we found that the wild strain was more toxic to RAW264.7 macrophages, and induced less the host cell apoptotic responses than the eha mutant did. Altogether, these data suggested that the Eha was required for the bacterial infection and the transcriptive regulation of the important virulence genes of E. tarda.

Eha, a transcriptional regulator of hemolytic activity of Edwardsiella tarda.

Hemolysis causes major symptoms such as the reddening skin and systemic hemorrhagic septicemia of diseased fish infected by Edwardsiella tarda. Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene in Escherichia coli K-12. In this study, we observed that the heterologous expression of the eha gene from E. tarda could confer hemolytic activity upon a hemolytic-silent E. coli strain. The transcription of clyA is positively controlled by the eha gene in E. tarda by RT-PCR. We cloned and purified Eha protein which had shown preferential binding ability to the clyA sequences in its promoter region, as evidenced by gel shift assay. The eha controls the transcriptional start predominantly at 72 bp upstream in the clyA promoter region, as determined by primer extension assays. We suggest that Eha protein is a new positive regulator found in E. tarda. In addition, we constructed the eha mutant and complementary strains of E. tarda. The hemolytic activity of the eha mutant was found to be attenuated compared with the wild-type strain. The complementary strains restored the hemolytic activity to levels between those of the wild type and the eha mutation. Our results indicate that the Eha protein is an important positive regulator in the hemolytic properties of E. tarda.