The Deletion Of Arca In Avian Pathogenic E.Coli And Research On The Mechanisms By Which Arca Contributes To Apec Pathogenicity

Avian pathogenic E.coli(APEC)cause one of the three most significant infectious diseases in the poultry industry and are also potential food-borne pathogens threating human health.In this study,The APEC TW-XM(02:K1)belonging to the phylogenetic E.coli reference(ECOR)group B2 was isolated from the brain of a duck which had septicemia and neurological symptoms.The strain was also found to cause serious meningitis in a neonatal rat model of human disease.Though mechanisms of APEC pathogenicity have been explored widely,there are still a lot of unknown potential virulence factors except classic ones.In this study,we show that ArcA(Aerobic Respiratory Control),a global regulator important for E.coli's adaptation from anaerobic to aerobic conditions and control of that bacterium's enzymatic defenses against ROS,is involved in the virulence of APEC.Deletion mutant of arcA were constructed using the bacteriophage lambda red recombinase system.A stable low-copy-number plasmid pGEN-MCS was used to construct a mutant complementation strain.To address the role of ArcA in APEC virulence,we compared the APEC XM wild type with its isogenic arcA deletion mutant strain for its ability to grow in vitro(LB)under aerobic and anaerobic conditions.The arcA mutant grew as well as the W T in LB medium under aerobic conditions and only slightly worse than the WT under anaerobic conditions.The virulence of WT and arcA mutant strain were then compared using a 7-day-old duck model.7-day-old ducks were divided into different groups according to our experiment,mortalities were recorded by oral challenge with various doses of viable bacteria.The ducks were observed for 7 days,mortality rates and fifty-percent lethal doses(LD50s)were calculated.The LD50 of WT was 3.43�104 CFU,and which of mutant strain was 1.49�106 CFU,and the LD50 of mutant complementation strain was 6.37�104 CFU.The result showed that deletion of arcA significantly attenuates virulence of APEC in the duck model.To further understand the mechanisms by which ArcA contributes to APEC pathogenicity,we used RNA-Seq to compare the transcriptomes of the APEC XM WT and its arcA mutant in duck serum and in LB broth.Results are reported here for the WT vs.arcA mutant comparison in duck serum.RNA-Seq analyses comparing the APEC wild type and the arcA mutant indicates that ArcA regulates the expression of 129 genes including genes involved in citrate transport and metabolism,flagella synthesis,and chemotaxis.To confirm the results from the RNA sequencing analyses,real-time quantitative reverse transcription-PCR was performed on the 10 genes,they were grxA,flgB,flgK,fliC,fliD,oppB,oppD,ynel,Ildr,glc.The expression of 7 of these genes was up-regulated and that of the other 3 was down-regulated,according to RNA sequencing analyses.The qPCR results were consistent with the results obtained from the RNA-seq analysis.However,for all genes,the qPCR results showed fold changes greater than those seen with the corresponding RNA-Seq analysis.Therefore,results of the qPCR analysis provided evidence that the transcriptional data obtained from RNA-Seq were reliable.From the result of RNA-Seq analysis,we did not find any classic virulence factors regulated by ArcA.In order to understand the mechanism of ArcA's role in pathogenesis,we selected glnL,glnG,flgB,flgK,fliC,fliD,motA,motB,cheA,cheW,citE,ciiF,citC.These genes involved in citrate transport and metabolism,flagella synthesis,and chemotaxis.Results of the qPCR analysis provided evidence that these genes were regulated by ArcA.Deletion mutant strains of cheA,cheAW,motAB,flgBCDEFGHIJK,fliCD,citCEFXG,glnLG were constructed using the bacteriophage lambda red recombinase system.To address the role of these genes in APEC virulence,WT and mutant strains were compared using a 7-day-old duck model.7-day-old ducks were divided into different groups according to our experiment,mortalities were recorded by oral challenge with various doses of viable bacteria.The ducks were observed for 12 days,mortality rates were calculated and compared by Fisher method.The result show that with an inoculation dose of 1�105 CFU,eight out of ten ducks inoculated with the WT strain died,while only three of the ten ducks inoculated with both cheA and motAB mutant strain died(p<0.05),suggesting that CheA and motA,motB increase the virulence of APEC.Surprisingly,the mutant with double deletion of cheA and cheW was as virulent as the wild type.And mutant strains of flgBCDEFGHIJK,fliCD,citCEFXG,glnLG,cheAW were as virulent as the wild type.Further study showed that the deletion of cheA,cheAWand motAB have no significant effect on APEC's ability to grow in vitro(LB)under aerobic and anaerobic conditions._The results indicate that ArcA's contribution to APEC pathogenicity is at least partially through regulation of the expression of motility and chemotaxis genes including cheA,motA and motB.Its the first report on the virulence role of ArcA in pathogenic E.coli.