Eha Regulated Edwardsiella Tarda To Adapt For Intracellular Survival

Edwardsiella tarda(Et)is an intracellular parasite,which belongs to EnterobacteriaceaeEdwairdsiella.Et haemolysin activator gene(eha)is an important transcriptional regulation gene,and can regulate Et to resist the oxidation and acidification pressure in macrophages,and contribute to the bacterial survival in macrophages.However,these mechanisms have not been fully elucidated.We try to detect the transcriptional levels of eha gene in different stress environment.Firstly,we amplified the promoter region of eha gene from ET13 genome as a template,constructed the promoter probe of pMP220-PehaLacZ vector,introduced the combination vector into eha mutant strain by electric shock and obtained the pMP220-PchaLacZ-Aeha strain.Then,we simulated the surviving environment in macrophages,and detected the ?-gal activities of pMP220-PehaLacZ-?eha strain in different oxidative and acidic conditions.Our results showed that the transcriptional levels of eha gene in the bacteria of log phase in treatment with pH6.3 and 0.10%H2O2 LB medium for 2h were higher than the ones in the other conditions,and the transcriptional level in treatment with pH6.3 LB medium was the highest.In order to detect the presence of self-regulation of eha gene,we compared the ?-gal activity of pMP220-PehaLacZ-ET 13 strains with the one of pMP220-PehaLacZ-Aeha strains,which were treated with pH 6.3 and 0.10%H2O2 LB medium for 2 h.We found that the ?-gal activity of pMP220-PehaLacZ-ET13 strains was significantly lower than the one of pMP220-PehaLacZ-?eha strains.The result indicated that there was a negative self-regulation on the expression of eha gene.We selected the acid conditions(pH6.3)to culture wild ET13 and its eha mutant strains,and to extract RNA of the two strains,in which the transcriptional level of eha gene was hightest.Their RNA purifies were so high to satisfy the requirements of RNA sequencing.The two kinds of RNA had been sent to to perform RNA-sequencing in Illumina HiSeqTM2000 of Beijing Genomics Institute.The sequence messages had been submitted to the database of NCBI Sequence Read Achieve(accession number:SRX 1898774),and evaluated the quality of sequence information.The mRNA of wild ET13 and eha mutant strains were randomly interrupted to construct two cDNA libraries.The randomness assessment showed that the two kinds of different length of clean reads were well-distributed in the reference genome of Et ATCC15947.Using SOAP aligner/SOAP2 software(http://www.ncbi.nlm.nih.gov/nuccore/),the two kinds of the clean reads were compared with the reference genomic sequence of Et ATCC15947.The coverage rates of the clean reads exceeding 90%of the two strains were more than 92%.Using RNA-Sequencing,we compared the transcripts of the two strains under acidic conditions,and screened 147 differentially expressed genes(DEGs)(| log2 Ratio | ?1),in which 113 genes were up-regulated and 34 genes were down-regulated.In addition,the 15 genes among the 147 DEGs were selected randomly to preform qRT-PCR.The results showed the two trends were strongly correlated,and verified the accuracy of RNA-Sequencing data.The 147 DEGs were divided into 25 functional categories via GO cluster analysis,which mainly involved in cellular process,localization,metabolic process,binding,catalytic activity,transporter activity,cell part and membrane.Through the KEGG Pathway analysis of the 147 DEGs,130 genes can be enriched into 55 pathways,including pathways such as amino acids,nucleotides and lipid metabolism,and iron transport.The main genesincluded two-component systems,ABC transports,microbial metabolism in diverse environments and biosynthesis of secondary metabolites etc.In order to find the target genes regulated immediately by Eha,we used commercial anti-Flag monoclonal antibody to enrich Eha-Flag protein.Firstly,we inserted Flag tag into the 3'end of eha gene by PCR,constructed the Eha-Flag expression vector pGEX-4T-ehaflag,and introduced it into eha mutant strain by electric shock?The experimental results of the cells infected by bacteria suggested that Eha-Flag fusion protein can restore the survival ability of eha mutant strain in macrophages.Then,we explored the ultrasound conditions of ChIP,used monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complex,removed the binding protein and purified the DNA fragment.We designed primers according to the DEGs of RNA-sequencing and identified the target genes combinated with Eha enriched by CHIP in qPCR.Finally,we got the ten target genes regulated immediately by Eha,including three genes related with amino acid metabolism,a rRNA-modified gene,a Fusaric acid resistance gene,a gene encoding the heat shock protein IbpB,a gene encoding the flagellin,a gene related with citrate metabolism,a gene encoding anaerobic C4 dicarboxylic acid transporter and a gene encoding hypothetical protein.Our study looked for the target genes regulated immediately by global transcriptive regulator Eha,found Eha function,understood the characteristic of the transcriptive regulatory mechanism of Et,and consummated the bacterial regulatory network.