The Role Of GltA And EpaPQR In Biofilm Formation Of Avian Pathogenic Escherichia Coli

Avian Pathogenic Escherichia coli(Avian Pathogenic Escherichia coli,APEC)causes persistent infections of poultry and causes multi-system diseases,which seriously endangers the development of the poultry industry.Biofilm is a kind of life phenomenon that bacteria adapt to the natural environment and are beneficial to survival.It is formed by the accumulation of microorganisms and their secretions.APEC's biofilm plays an important role in the process of infection and disease.Escherichia coli Type III Secretion System 2,ETT2 is involved in the pathogenic effect of APEC and various cellular behaviors,and participates in the formation of APEC biofilms,but the role of ETT2 in the formation of biofilms still not clear.In addition,APEC's biofilm regulatory network is complex and requires the coordinated regulation of numerous systems.Therefore,searching for various unknown related factors in the formation of APEC biofilm and analyzing their mechanism of action is of great significance for the prevention and control of APEC infection and disease.This study was based on the Tn-5 transposon mutation library platform to screen APEC biofilm formation related genes,and analyzed the biological characteristics of the citrate synthase gene glt A,which has not been reported to be related to biofilm formation.Regulating the formation of APEC biofilm.After that,the 5 hypothetical structural genes in the epa gene cluster in ETT2 were analyzed by bioinformatics,and the epa PQR gene that may have the function of the basement intima of the injection device was obtained by bioinformatics analysis,and the deletion and reverting strains of the epa PQR gene were constructed.Analyze its influence on APEC biofilm formation and pathogenicity.The main research contents and results of this study are as follows:1.Screening of genes related to biofilm formation based on Tn-5transposon mutation libraryIn the Tn-5 transposon mutation library constructed by our laboratory with the AE81?ETT2 deletion strain,strains with defects in biofilm formation were screened by crystal violet staining.The screened strains with defects in biofilm formation are determined by TAIL-PCR and multiple sequence alignment techniques to determine the gene at the transposon insertion site,and analyze the regularity of transposon insertion and the regulatory effects of different functional genes on APEC biofilm formation.And use red homologous recombination technology to construct and screen the deletion strains of biofilm-related genes,and verify the correlation of biofilm formation by crystal violet staining.The results showed that the AE81 genome-wide modeling results showed that Tn-5 was randomly inserted into the AE81 genome;a total of 14 strains with significant differences in biofilm formation defects(P<0.05)were screened through the mutation library,and their biofilms were reduced.The rate is between 34.0% and 80.5%;TAIL-PCR and multiple sequence comparison results show that 14 strains contain 7 different gene insertion sites,including 2 flagella genes(fli S and fli D),and 2 curli pili Genes(csg A and csg D),type? fimbriae gene(fim A),ABC transporter gene(mla A)and 1 coenzyme gene(glt A);the red homologous recombination technology was used to successfully construct the abovementioned 7 transposon insert genes The biofilm test results of the deletion strains showed that the biofilm formation ability of the 7 deletion strains was significantly reduced(P<0.05),while the flagella gene and fimbriae gene had a stronger ability to reduce APEC biofilm formation than other gene-deficient strains;AE81 genome-wide modeling results show that Tn-5 is randomly inserted into the genome of AE81;the deletion strains of 8transposon insertion genes constructed in AE81 and the biofilm formation ability test results prove our screening and the correctness of the analysis.2.Citrate synthase gene glt A participates in the regulation of APEC biofilm formation mechanismBased on the research content I screened APEC biofilm formation-related genes,and explore the mechanisms involved in regulating biofilm formation.The growth curve,motility and rdar morphology of the selected gene-deficient strains were analyzed,and the reported biofilm-related gene regulatory pathways were verified by fluorescence quantitative experiments.The mechanism by which citrate synthase glt A participates in APEC biofilm formation has not been reported,so this gene is focused on research.The results of growth curve determination showed that the growth ability of the 7 strains lacking transposon insertion genes was not significantly different(P>0.05);the exercise test results showed that the exercise ability of the three deleted strains of AE81?fli S,AE81?fli D and AE81?glt A decreased;The results of the rdar morphology test showed that AE81?csg A exhibited pas(pink and smooth)colony morphology,indicating that it lacks fimbriae production and weak cellulose expression;AE81?csg D exhibits saw(smooth and white)colony morphology,indicating that it lacks fimbriae The other five deleted strains and AE81 showed rdar(red morphology,dry and rough)morphology,indicating that both fimbriae and cellulose were expressed normally.The fluorescence quantitative PCR results showed that the flagella gene-deficient strain AE81?fli S,The transcription level of the flagellar master operon flh D/C in AE81?fli D was significantly down-regulated,the transcription level of the operon csg D in the fimbriae gene-deficient strain AE81?csg A was significantly downregulated,and the transcription of bcs A,mlr A,adr A,flh D,flh C biofilm-related genes in the AE81?glt A-deficient strain The level was significantly down-regulated(P<0.05).Therefore,glt A may be involved in regulating the formation of biofilms by changing the output of bacterial cellulose,fimbriae and flagellin.3.the study of the effect of epa PQR gene on the formation and pathogenicity of APEC biofilmFive assumed functional genes of ETT2 epa PQR gene cluster were analyzed by bioinformatics,and three structural genes epa PQR were screened.Based on CRISPR-CAS9 gene editing technology,the deletion and recovery strains of epa PQR gene were constructed,and the effects of membrane forming ability and pathogenicity of epa PQR gene were studied.The results showed that the growth ability of epa PQR gene was not significantly changed(P> 0.05);the biofilm formation ability was not significantly changed(P > 0.05);the sensitivity of 12 drugs was not significantly changed;but after the deletion of epa PQR gene,its motor ability decreased significantly(P < 0.05),and the flagella number was significantly reduced by fluorescence setting The results showed that the transcription level of T3 SS and the flagellum output protein gene were significantly decreased(P < 0.05).The results showed that the ability of antiserum bactericidal activity was significantly increased after the deletion of epa PQR gene(P < 0.05);the pathogenicity test showed that the colonization ability of the missing strain AE81?epa PQR in different organs in the chicks was significantly reduced compared with the wild ones(P < 0.05).In conclusion,eight membrane forming genes with different functions were screened through the trans transposon Library of Tn-5.Eight missing strains were successfully constructed by red homologous recombination technology.The regulatory mechanism of these known and unreported glt A genes related to the biofilm formation of E.coli was explored.The epa PQR gene of ETT2 gene failed to change the ability of APEC biofilm formation,but participated in regulating the flagella formation,antiserum bactericidal ability and the ability of tissue and organ colonization in vivo,which affected the pathogenicity of bacteria.