Role Of Transcription Regulator EivF In The Pathogenicity Of Avain Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)as a common pathogenic bacteria often interacts with other pathogenic bacteria,causing colibacilosis in poultry and even cause mortality in poultry.There are many complex serotype and numerous pathogenic factors which are the major problems in the control and prevention of APEC.Therefore,APEC has great importance in field of research regarding to public health prevention and control(zoonotic point of view).Escherichia coli Type III Secretion System 2(ETT2)is an important virulence effector of APEC and it is also found in the majority strains of E.coli.It's also play an important role in pathogenic process in the bacterial toxin secretion and transportation.The transcription factor eivF gene belongs to the ETT2 virulence island.At present,the eivF gene has only studied in Enterohemorrhagic Escherichia coli and found that it can regulate Locus of Enterocyte Effacement(LEE)virulence secretion and participate in the process of bacterial adhesion.It has not been studied in APEC and other E.coli.In this study,we deleted the gene eivF from the wild strain of APEC AE81 and named as mutant strain which denoted by AE81?eivF and also construct another strain called complementary strain denoted by AE81?eivF-comp.The construction of mutant and complementary strain from a wild strain of APEC ETT2 is done for the evaluation of their impact on biological characteristics of APEC,and RNA-Seq technology was used to screen the significantly expressed function of gene eivF,and it was clarified that eivF has potential power in transcriptional regulation.The study on pathogenic effect of eivF will provide the fruitful information for further research in future on the pathogenic mechanism of ETT2 and the pathogenic mechanism of APEC.The contents and results of this research are summarized as follows:1.Construction of mutant strain AE81?eivF and complementary strain AE81?eivF-comp from the wild strain of AE81 and their effects on biological characteristicsBy help of PCR,using ampicillin and chloramphenicol as screening agent which showed that the APEC strain AE81 contained the target gene eivF.The APEC eivF gene mutant was carried out by using Red homologous recombination technology,and AE81?eivF-comp strain was constructed using the low copy of plasmid p STV28.Thegrowth curve measurement results showed that there was no significant difference in the growth performance between mutant strain AE81?eivF and wild strain AE81.The antibiotic sensitivity test showed that there also no difference between the inhibitory zone of mutant strain AE81?eivF and wild strain AE81.The motility test results showed that the wild strain AE81 formed a larger movement circle,and the mutant strain AE81?eivF movement circle became significantly smaller.After mutant eivF,its exercise ability weakened,and the complementary strain AE81?eivF-comp basically recovered to the wild strain state,forming a larger movement circle.The wild plant AE81 flagella was slender and numerous,the mutant strain AE81?eivF have low motility because the number of flagellae of mutant strain AE81?eivF was significantly reduced,and the complementary strain AE81?eivF-comp had more flagella which was observed by Transmission Electron Microscopy.The results of biofilm showed that the wild strain AE81 has a strong film-forming ability,can form a complete biofilm,and the membrane surface is smooth and flat.The biofilm of the mutant strain AE81?eivF is intact and the membrane surface is obviously wrinkled.The surface of the complementary strain AE81?eivF-comp membrane was slightly wrinkled.Through scanning electron microscopy,it was found that the wild AE81 biofilm was smooth and dense,and the adhesion between bacteria was tight.The spatial structure of the mutant strain AE81?eivF biofilm is more complex and three-dimensional,with holes similar to the three-dimensional tower structure.The biofilm of the complementary strain AE81?eivF-comp is flat and dense with no pore structure.The results of acid tolerance and hydrogen peroxide sensitivity test showed that the acid tolerance and hydrogen peroxide resistance of mutant strain AE81?eivF was significantly reduced as compared to the wild strain AE81(P <0.05).2.RNA-Seq analysis of APEC wild strain and eivF mutant strainThe AE81?eivF differentially expressed genes of mutant strain AE81?eivF by RNA-Seq technology.The results showed that the mutant strain AE81?eivF had 576 significantly differentially expressed genes,including 368 are up-regulated genes and 208 are down-regulated genes.The functions involved mainly include cellular processes,metabolic processes,By Gene Ontology(GO)analysis we found that 38 functions were annotated.The main functions were cellular processes,metabolic processes,positioning;cells and cell components,membranes and membrane components;catalytic activity,binding,transcriptional activity,etc.KEGG(Kyoto encyclopedia of genes and genomes)pathway analysis showed that the main pathways for enrichment of differential genes wereflagella assembly,microbial metabolism in adverse environments,ABC transporter,antibiotic biosynthesis,two-component system,carbon metabolism,quorum sensing and biofilm formation-E.coli,etc.Combined with the content of the research,there are significant differences in the biological phenotypes of flagella and biofilm.From the transcriptome data,the differential genes related to flagella assembly and biofilm formation were screened.It was found that the differential genes of flagella mainly involved flagella level II and level III regulatory genes,such as flg B,flg C,flg D and mot A,etc.Biofilm differential genes mainly involve transcriptional regulatory genes related to biofilm formation,such as mcb R,csg D,and mlr A.Some differentially expressed genes were screened for reverse transcription real-time quantitative PCR detection.The results showed that their transcription level trends were consistent with transcriptome data.3.The effect of eivF mutant on the pathogenicity of APECThe pathogenicity of APEC were checked by hemolysis test,serum sterilization,cell adhesion and by injecting it in in-vivo after the mutant of eivF.The results of hemolysis test showed that there was no significant change in the hemolysis status of mutant strain AE81?eivF.The results of serum sterilization test were also showed that there were no significant change of this mutant strain AE81?eivF strain at the level of 10 %,20 %,30 %and 40 % SPF serum in PBS,but at the level of 50 % serum concentration(P<0.05)the ability of mutant strain AE81?eivF has been increased for serum resistance.Chicken embryo fibroblast(DF-1)adhesion test results showed that the mutant strain AE81?eivF has greater ability of bacterial adhesion cells(P<0.05)as compared to wild strain AE81 which was detected by RT-q PCR.It is also found that the deletion of tsh,bcf A and ibe A gene also increased their cell adhesion properties(P<0.05)but there was no significant change in fim C gene deleted strain.By the in-vivo results we found that after the deletion of the eivF gene,the number of bacterial colonies in the tissues of poultry heart,liver,spleen and lungs were increased significantly(P<0.05).The above results showed that eivF is involved in the pathogenic role of avian pathogenic E.coli.In this study we successfully constructed strain the eivF mutant and complementary strains.After the mutant of eivF the bacterial motility,acid resistance and hydrogen peroxide sensitivity were significantly reduced.On other hand the biofilm formation,antiserum resistance ability,cell adhesion and in-vivo bacterial load was significantly increased,but there was no significant difference in growth performance and hemolysis.The eivF also affects the transcription level of flagella,biofilm and adhesion,and all thesecharacters are related to bacterial pathogenicity.It indicates that eivF is involved in the regulation of APEC biological phenotype and pathogenesis,which may affect the pathogenesis of APEC by regulating flagella,biofilm and adhesion genes.