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-P_targetlacZ and pBD-P_ehalacZ 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 β-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是Tarta Edwardsiella（E. tarda）的毒力调节剂）。本研究检查了Eha如何调节其靶基因以影响细胞内细菌的存活。我们构建了一个报告基因，构建了一个在其C末端标记了带有标记表位的Eha的pGEX-4T-ehaflag质粒，并将该质粒引入到一个eha突变ET13菌株中，从而获得了一个Cehaflag菌株。EhaFlag融合蛋白的表达和活性通过Western印迹和细胞内存活实验恢复了巨噬细胞中Cehaflag的野生型存活。我们使用单克隆抗-Flag抗体使用彩色免疫沉淀（ChIP）沉淀EhaFlag-DNA复合物。然后，我们基于从RNA测序中鉴定到的差异表达基因设计引物，并通过qPCR鉴定了十个与Eha相互作用的基因。我们扩增了这10个基因的启动子区域，通过PCR从ET13菌株获得eha基因，构建了pBD-P_靶标lacZ和pBD-P _eha lacZ质粒。通过比较Eha基因的β-半乳糖苷酶活性，eha基因直接和正向调节了这些靶基因，并被Eha在E. tarda中负向自动调节。这些靶基因分布在与细菌生长，运动和对H ₂ O _2的抗性有关的类别中或酸。我们进一步构建了ETATCC_RS15225突变体（△dcuA1），ETATCC_ RS14855突变体（△flgK）和ETATCC_RS07650突变体（△tnaA），以及△eha-tnaA和△eha-flgK的部分互补菌株以及C△flgK和CΔtnaA的互补菌株。ETATCC_RS15225基因可能通过SDS-PAGE和RT-PCR在外膜上编码转运蛋白DcuA1。ETATCC _RS14855基因可能编码FlgK蛋白并影响细菌运动。ETATCC_RS07650基因编码色氨酸酶，该酶影响巨噬细胞内细菌的存活。在上述菌株的协助下，我们的结果表明eha基因能够调节ETATCC_RS15225基因表达其外膜蛋白DcuA1，ETATCC_RS14855基因可控制鞭毛运动，而ETATCC_RS07650基因可影响巨噬细胞内细菌的存活。结合以上三个基因的其他功能，我们的结果表明Eha直接调节靶基因以影响大肠杆菌在细胞内存活。