Research On The Pathogenesis Of Pathogenic Escherichia Coli By Global Regulators ArcA And FNR

Pathogenic Escherichia.coli(E.coli)can be classified as Extraintestinal Pathogenic Escherichia coli(ExPEC)and Intestinal Pathogenic Escherichia coli(IPEC)according to pathogenicity and pathogenesis.Enteropathogenic E.coli mainly includes Avian Pathogenic E.coli(APEC),Uropathogenic Pathogenic E.coli(UPEC)and Neonatal Meningitis.E.coli,NMEC).Avian pathogenic E.coli(APEC)is one of the most important infectious pathogens in the poultry industry and a potential foodborne pathogen.APEC causes extraintestinal infections—primarily respiratory infections,pericarditis,and septicaemia of poultry.Human ExPEC can cause urinary tract infections or neonatal sepsis and meningitis.Enteropathogenic Escherichia coli is divided into six categories based on its pathogenic characteristics:Enterotoxigenic E.coli(ETEC),enteropathogenic E.coli(EPEC),Enterohemorrhagic E.coli(EHEC),Enteroaggregative E.coli(EAEC),Enteroinvasive E.coli(EIEC),and Diffuse Adhesive E.coli(DAEC).Their common characteristic is that they cause severe diarrhea by secreting different toxins on human or animal intestinal epithelial cells.The most serious clinical hazards are ETEC,EHEC and EPEC,which cause serious economic losses to animal husbandry and aquaculture every year.ExPEC and IPEC infections are serious threats to the health of humans and animals.Strengthening research on the prevention and control of ExPEC and IPEC is of high importance for aquaculture and public health.The current strategies of prevention and control of ExPEC and IPEC is heavily relying on the use of antibiotics,which has caused the emergence of drug-resistant strains and antibiotic residues.Therefore,there is an urgent need to develop new alternative strategies.However,the pathogenesis and its regulatory mechanisms of ExPEC and IPEC are not fully understood,which hinders the developmentof novel strategies to control the diseases caused by ExPEC and IPEC.Arc A and FNR,as global regulators,play important roles in regulating E.coli metabolism adaptation to aerobic and/or anaerobic conditions.The role of Arc A and FNR in the pathogenesis of ExPEC and IPEC has been hinted in several previous studies,but is not well defined.In this study,the transcriptomes of ExPEC 02:K1 during growth in LB and serum were compared.The regulatory mechanisms(ArcA and FNR)underlying differential expression of metabolism and virulence genes were elucidated.At the same time,we carried out whole genome sequencing of 95 E.coli strains,analyzed their drug resistance and strain typing,and preliminary explored the regulation of ETEC pathogenesis by ArcA and FNR.1 Transcriptome Sequencing Analysis Screening ExPEC for Host Differentially Expressed GenesWe compared differentially expressed genes of ExPEC in response to serum and LB under microaerobic conditions by using transcriptome sequencing.Using a threshold false-discovery rate of 10%and comparisons with q values of 0.10,a total of 427 genes were found to be significantly upregulated and with 390 genes downregulated under conditions simulating growth in vivo conditions.These genes could be classified into four categories:mobility and chemotaxis related genes(motAB,cheAW,flgB-M and fliA-S):nitrogen and various amino acid metabolism related genes;carbon center metabolic pathway and respiratory chain related genes(citCDEFXG,citAB,ybdS and frdABCD);and genes related to E.coli exopolysaccharide(ECP)biosynthesis(LPS biosynthesis genes,enterobacterial common antigen K-capsule peptidoglycan,colanic acid,Yjb exopolysaccharides and D-ribose transport).These results revealed the ExPEC' adaptive response to systemic infection.2 ArcA Controls Chemotaxis,Motility and Metabolism Contributing to the Pathogenicity of ExPECStudies have shown that global regulators ArcA regulate the expression of a large number of metabolically related genes in E.coli under anaerobic conditions.However,whether ArcA play an important role in the pathogenesis of ExPEC remains undetermined and their underlying molecular regulatory mechanisms require further studies.The resultsof real-time quantitative PCR confirmed the results of transcritomic sequencing that the expression levels of cheA,cheW,cheA&W,motA,motB,motA&B in ?arcA strains were significantly lower than those in wild strains under hypoxic conditions,indicating that ArcA directly or indirectly regulates the expression of these genes.Reverse transcription PCR confirmed that motA,motB,cheA,and cheW share one promoter and EMSA results showed that ArcA protein can directly bind to motA promoter.Altogether,our results demonstrated that ArcA directly controls chemotaxis and motility contributing to the pathogenicity of ExPEC.Although the two-component system CitA/CitB has been reported to be involved in the regulation of bacterial citrate fermentation genes,the regulatory mechanisms underlying its function under anaerobic conditions have not been fully understood.Our transcriptomic studies showed that the expression levels of citrate fermentation genes,including citC,citE,and citF,were significantly down-regulated in ?arcA mutant compared to those in wild-type(WT)strain when cultured in serum under microaerobic conditions.The results of qRT-PCR confirmed that the citC expression was significantly downregulated in AarcA strains lower in serum under microaerobic conditions.Further studies on citrate metabolism-related genes revealed that ArcA can directly regulate the expression of citrate fermentation genes,and indirectly regulate the expression of citrate fermentation genes through the CitA/CitB two component system.Animal experiments showed that the deletion arcA,motAB and che.4 significantly reduced the virulence of ExPEC,but deletion of citCDEFXG had no significant effect on ExPEC virulence.However,the AcitCDEFXG mutant grew slower than WT during the lag and log phases when cultured in serum,indicating that citrate citCDEFXG increases APEC's growth in duck serum under microaerobic conditions.In conclusion,we identified an additional regulator,ArcA,which indirectly regulate the expression of chemotaxis,motility and citrate fermentation genes.3 FNR Increases Antiserum Capacity of Bacteria by Regulating ExPEC Citrate Fermentation Genes and ECP GenesBioinformtcics analysis predicted that the upregulated genes involved in citric acid metabolism and Extra-cytoplasmic polysaccharide(ECPs)biosynthesis genes in ExPEC in serum and low oxygen tension could be regulated by oxygen sensor FNR.Indeed,deletion of fnr gene significantly reduced the utilization of citrate of ExPEC.Subsequent assays confirmed that FNR directly control the expression of citrate fermentation genes,but also indirectly affect the expression of citrate fermentation genes by regulating the CitA/CitB system.In addition,qRT-PCR showed deletion of fnr significantly decreased the expression of 11 selected ECP biosynthesis genes and reintroduction fnr into the mutant recovered their expression to wild type's level,confirming that Fnr directly or indirectly regulate the expression of ECPs biosynthetic genes.EMSA demonstrated that FNR could shift wza and kpsM DNA promoter and upstream regions with FNR-binding site but not without FNR-binding site,suggesting that FNR could directly regulate their expression.Furthermore,results showed FNR indirectly modulated the expression of colanic acid,Yjb exopolysaccharides and peptidoglycan biosynthesis genes also via the Rcs signaling system.Animal tests showed that fnr,Res signaling system and ECP biosynthetic genes contribute to the pathogenicity of ExPEC.These findings suggest that the global regulator FNR could affect both metabolisn and ExPEC's serum resistance,therefore contribute to ExPEC's virulence.4 Molecular mechanisms of ETEC virulence genes by ArcA and FNREnterotoxigenic Escherichia coli cause severe diarrhea,fever and acute death in humans or animals.Therefore,studies on the resistance,epidemiology and pathogenic molecular mechanisms of ETEC can help to prevent and control ETEC and reduce the harm caused by ETEC infection.In the present study,95 E.coli strains isolated from intestinal contents or fecal samples of diarrheic piglets in 13 states were determined for phenotypic antimicrobial susceptibility to 11 antibiotics,the resistance genotypes and virulence genes by whole genome sequencing.One representaitve strain was selected for expression study.The expression levels of adhesin genes fedA,eaeH,enterotoxin genes eltA,st2,and hemolysin genes hlyC,hlyE at different time points after ETEC interaction with IPEC-J2 cell was determined.These results showed that the expression of these genes was increased to a maximum after interact with cells for 1 h.The expression levels of these genes in arcA and fnr mutant were down-regulated,and further studies confirmed that ArcA and FNR can directly regulate the expression of these genes.Adhesion test results showed that deletion of arcA and fnr significantly down-regulated the adhesion of ETEC to IPEC-J2 cells.These results indicate that ArcA and FNR contribute to the pathogenecity of ETEC by directly regulating virulence genes such as fedA,eaeH,eltA,st2,hlyC and hlyE.