The Role Of MotA/B And CheA/Y Of Avian Pathogenic Escherichia Coli In PhoP/Q Regulation Of Flagellum T3SS Pathway

Avian Pathogenic Escherichia coli(APEC)is an important causative agent for parenteral infection in poultry,and the pathogenicity of APEC is mainly determined by the expression of its virulence genes.phoP/Q is one of the most important regulatory elements in avian pathogenic Escherichia coli,which can regulate many components affecting bacteria.Flagella is an important movement and pathogenic component of Escherichia coli.Type ? secretion system(T3SS)is the core and foundation of bacterial flagella.motA and motB are transmembrane proteins that control the movement of flagella and affect the direction and progression of bacterial movement.cheA is a histidine kinase involved in the signaling of chemotactic receptors.cheY is a diffuse response regulatory protein that can be phosphorylated by cheA and can alter the motor of flagella.Both groups of two-component systems are closely related to flagella,and the specific mechanism of the regulation of phoP/Q on flagella has not been reported in the literature.Therefore,the two groups of two components were selected as the entry point for studying the mechanism of the regulation of phoP/Q on flagellaThis study aimed to explore the link between the two-component system phoP/Q in APEC and the flagellar type ? secretion system(flagellate T3SS).AE17,a wild type strain of APEC,was selected as the research object,and the distribution of flagellar T3SS and its related two-component genes was statistically analyzed by PCR.The link between phoP/Q and flagella T3SS was predicted by software.Then,four genes,which may be intermediate regulators,were knocked out,such as motA,motB,cheA,and cheY,to obtain four single gene deletion strains,and the biological characteristics of the gene deletion strains were studied.The relationship between motA/B,cheA/Y,phoP/Q and flagella T3SS was further verified by real-time PCR and ?-galactosidase assay.The specific research contents and results are as follows:1.Effects of two-component systems of motA/B and cheA/Y on biological characteristics in avian pathogenic Escherichia coliAn O2 serotype clinical wild strain avian pathogenic Escherichia coli AE17 isolated and preserved in the laboratory was used as an experimental strain.Identification and deletion of the target genes motA,motB,cheA,and cheY were performed.As a result,four single gene deletion strains(?motA,?motB,?cheA,and ?cheY)were successfully constructed.vThereafter,the effects of motA,motB,cheA,and cheY gene deletion strains on APEC growth characteristics,exercise characteristics,and biofilm formation were examined by measurement of growth curve,detection of motility,and biofilm formation ability.Biofilm experiments showed that the deletion of motA and cheA genes accelerated the growth of AE17 strain,while the deletion of cheY gene significantly inhibited the growth of AE17 strain.The experimental results of exercise capacity showed that the motility of ?motB was significantly weakened compared with the original strain,while the motility of ?cheY was significantly enhanced.The biofilm test results showed that compared with the wild strain AE17,the biofilm formation ability of the four strains was reduced,but no significant difference was observed(p<0.05)?2.Distribution and pathway prediction of motA/B,cheA/Y,phoP/Q and flagellum T3SS genes in avian pathogenic Escherichia coliUsing 39 clinically isolated wild-type avian pathogenic Escherichia coli strains stored in the laboratory as templates,28 flagella T3SS related genes and 7 two-component system gene primers were designed to detect gene distribution.Review the relevant literature and use the protein interaction network database "string" to predict the relationship between phoP/Q,flagellar T3SS-related genes and genes near the locus.The results showed that 28 flagella T3SS genes and their related 7 two-component system genes had higher detection rates in different serotypes of APEC.PhoP/Q and flagellum T3SS are closely related to motA/B and cheA/Y,respectively.PhoP/Q may indirectly regulate flagellum T3SS by two-component genes(motA/B and cheA/Y)around flagellum T3SS gene cluster.3.Analysis of correlation between phoP/Q,motA/B,cheA/Y genes and flagellum T3SS in avian pathogenic Escherichia coliTotal RNA of AE17 strain and ?phoP,?phoQ and ?phoP/Q deletion strains previously constructed in the laboratory were extracted,and then reverse transcribed to obtain cDNA,and the transcription levels of related genes of motA/B,cheA/Y and 28 flagellum T3SS were detected The results showed that the single deletion and double deletion of phoP and phoQ genes showed significant differences in the transcription levels of at least one gene in the two-component system genes(p<0.01),which would significantly change the transcription level of more than half of the 28 flagellar T3SS genes.And the deletion of these two genes will cause significant changes in the transcription levels of motA/B and cheA/Y genes.(p<0.01)Six(pRCL-flhEAB,pRCL-cheY,pRCL-motBA,pRCL-fliD,pRCL-flgH,pRCL-phoPQ)?-galactylase promoter were constructed in this experiment by analyzing and screening fluorescence quantitative PCR data.Recombinant plasmid.Six plasmids were introduced into AE17 strains,?phoP,?phoQ,?motA,?motB,?cheA,and ?cheY,and?-galactosinase activity was detected.The results showed that motA and motB and cheY proteins had significant positive feedback regulation on the transcriptional initiation of phoP/phoQ gene,PhoQ and cheA and cheY proteins had significant positive feedback regulation on the transcriptional initiation of motA/motB gene,PhoP and PhoQ and cheA proteins had significant negative feedback regulation on the transcriptional initiation of fliD gene,cheY proteins had significant positive feedback regulation on the transcriptional initiation of fliD gene,phoP and phoQ and cheA and proteins had significant positive feedback regulation on the transcriptional initiation of flhE/flhB/flhA gene,and motB protein had significant negative feedback regulation on the transcriptional initiation of flhE/fhB/flhA gene.Through the above experiments,motA,motB,cheA,and cheY single gene deletion strains were successfully constructed.These four genes can affect the growth and movement of bacteria,which may be related to flagellar formation.motA/B,phoP/Q,cheY/A and flagella T3SS genes have higher detection and conservation in APEC.The two-component system genes of motA/B,cheY/A,and phoP/Q can regulate most flagella T3SS genes,and the three pairs of two-component system can also regulate each other.It is predicted by software and experiments that phoP/Q may regulate flagella T3SS indirectly by regulating motA/B and cheY/A.