Effects Of C-di-GMP Metabolic Genes And Quorum Sensing System On Biological Characteristics Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)is one of the main pathogens of poultry,which seriously affects the development of poultry breeding.APEC can enter the poultry through the respiratory tract and adhere to and colonize the lower respiratory tract.The toxins can cause acute sepsis and multiple organ damage in poultry.Due to the large number of APEC serotypes and complex drug resistance mechanisms,it is difficult to prevent and control APEC disease.Biofilms have important regulatory effects on the pathogenicity of APEC.Biofilm(BF)is a bacterial adhesion to the surface of a substance and is encapsulated by an extracellular matrix to form a multicellular population morphology with a tight structure.The formation of BF is regulated by many factors,among which the quorum sensing system and c-di-GMP are the main regulatory mechanism.In this study,we further studied how quorum sensing systems and c-di-GMP regulate the formation of APEC biofilms,which in turn affects their biological characteristics.In this study,the c-di-GMP metabolic genes involved in APEC biofilm formation were screened,and the effects of quorum sensing system on APEC biofilm formation were also studied.The regulation mechanism of c-di-GMP and quorum sensing regulation of APEC biofilm formation was preliminarily studied.It provides a reference for the prevention and control of APEC from the perspective of c-di-GMP and quorum sensing systems.1.Functional verification of c-di-GMP metabolic gene yegE and its effect on the biological characteristics of APEC.c-di-GMP is a universal second messenger that regulates various biological characteristics of bacteria such as virulence,biofilm formation,motility,drug resistance,cell morphology and cycle.The preliminary results of our laboratory showed that 35 c-di-GMP metabolic genes were identified in the APEC clinical isolate strain DE17.By constructing different c-di-GMP metabolic gene mutant strains,the yegE gene was screened to regulate the biofilm formation of DE17.Analysis of the amino acid sequence of YegE indicated that it contained a GGEEF domain.By constructing the pET28b-yegE expression vector,it was electrotransformed into the strain BL21(DE3)that containing the amcyan and turbo rfp fluorescent plasmid pRP0122-atr,which inserted of 3 natural tandem riboswitch between the two fluorescent genes.After induction by 0.5 mM IPTG for 18 h,amcyan and turbo rfp fluorescent was observed under the fluorescence microscope,and the color of bacterial broth showed orange.Induced by no or 0.05 mM IPTG,it only showed amcyan fluorescent under the fluorescence microscope,and the bacterial broth color was green,indicating that the gene yegE has a c-di-GMP synthetase functionThe Red recombination system was used to successfully obtain the mutant strain DE17AyegE.The study results of biological characteristics showed that,compared with wild-type,yegE significantly reduced the biofilm formation ability of DE17(p<0.01),and enhanced the swarming ability(p<0.05).yegE also altered its rdar morphology.However,it has no significant effect on its growth performance and drug resistance.The results of real-time PCR showed that,compared with the wild-type strain,the transcriptional level of the biofilm formation-associated genes self recognizing antigen 43 autotransporter(agn43)from the mutant strain was significantly reduced by about 5-fold,and the extracellular polysaccharide synthesis subunitpgaD was up-regulated by 2.2-fold.The flagella transcriptional regulatory factor flhC and flagellin-synthesizing protein factor flhA was up-regulated by 3-fold and 9.6-fold,respectively.There was no change in the transcription level of the extracellular polysaccharide synthesis subunit pgaA,the polysaccharide biosynthesis/export protein wza and the flagellar motor switch protein fliM,The results of this study indicate that the yegE acts as a c-di-GMP synthetase gene involved in the regulation of biofilm formation in DE17.This study provides a basis for further development of the molecular mechanism of c-di-GMP metabolic gene regulation of biofilm formation in APEC.2.The regulation of the biological characteristics of APEC by type I quorum sensing systemStudies have shown that in Gram-negative bacteria like Escherichia coli and Salmonella,the type I quorum sensing signal molecule AHL receptor SdiA(Suppressor of division inhibitor,SdiA)is present,but lack of the AHLs signaling molecule synthesis gene.In order to study the regulation of APEC by type I quorum sensing system,in this study,AHLs synthetic gene lasl from Pseudomonas aeruginosa with a over-expressing pasmid was transformed into APEC strain DE17,and constructed a type I quorum sensing system in DE17 to study wild-typeand recombinant strains(DE17).-lasI)changes in biological characteristics.The results showed that compared with the wild-type strain DE17,the biofilm formation ability and motility of the recombinant strain(DE17-lasI)were significantly decreased(p<0.01),and its rdar morphology was changed,but its growth characteristics and drug resistance were not significant changed.The results of real-time PCR showed that compared with wild-type strain DE17,the transcription level of AHLs receptor gene sdiA in recombination strain DE17-lasI was up-regulated by about 9-fold;The transcription level of self recognise antigen 43(agn43)which related to biofilm formation was down-regulated by about 4-fold;The motility related gene flagellin transcriptional activator flhC was down-regulated by 2.5-fold;The virulence-associated pilus adhesin subunit fimH was up-regulated by about 60-fold,while the transcriptional levels of virulence genes such as serum-stimulating protein iss and outer membrane porin protein ompA were down-regulated by 21.7-fole and 4.4-fold,respectively.This study showed that the type I quorum sensing system regulates the biofilm formation of DE17 and participates in the regulation of some biological characteristics in DE17.This study provides a reference for further research on the regulation of APEC by the type I quorum sensing system.3.The LuxS/AI-2 quorum sensing system affect the formation biofilm of APEC by regulating the c-di-GMP metabolic gene yeaP.Previous studies in our laboratory have shown that both the LuxS/AI-2 quorum sensing system and c-di-GMP can regulate the biofilm formation in APEC.To study whether the LuxS/AI-2 quorum sensing system can affect the biofilm formation in APEC by regulating c-di-GMP metabolic genes,in this study,we designed a double gene mutant strain which was luxS and different c-di-GMP metabolic genes that affects the biofilm formation in DE17,and identified c-di-GMP metabolic genes regulated by LuxS/AI-2 quorum sensing system.Then further study about the mechanism of LuxS/AI-2 dffect biofilm formation by regulating the c-di-GMP metabolic genes.The results showed that the luxS gene and yeaP gene did not affect the biofilm formation in DE17.Furthermore,comparing with the wild strain DE1 7,in the double gene mutant strain DE17?1uxSAyeaP,its biofilm formation ability was significantly improved(p<0.01),and the motility was significantly reduced(p<0.05),the rdar morphotype was also changed,but its growth performance and drug resistance did not be affect.The results of the autolysis test and the extracellular eDNA quantification showed no difference among the double gene mutant strain,the single gene mutant strain and the wild strain.Transmission electron microscopy of the double gene mutant strain showed that the number of pilus of the double gene mutant strain decreased and the number of flagella increased.Laser scanning confocal microscopy showed that the number of bacteria in the biofilm of the double gene mutant strain was much more than the wild-type strain and the single gene mutant strain.The results of real-time PCR showed that the transcription levels of agn43,wza and bcsA of the double gene mutant strain were up-regulated by 5.7-fold,22.5-fold and 12.6-fold,respectively,compared with the wild-type strain.The transcription levels of pgaD,csgD and fimC were down-regulated by 1.7-fold,7.6-fold and 2.4-fold,respectively.Serum bactericidal assays,distribution in vivo,and DF-1 cell adhesion and invasion assays were used to assess virulence changes in mutant strains and wild-type strain.The results showed that the absence of yeaP gene had no significant effect on virulence of DE17.However,comparing with wild-type and single gene mutant strains,in high concerntration of complement,the number of double gene mutant strain DE17?luxS?yeaP of survival bacteria was significantly decreased,and it has a concerntration dependence.In addition,comparing with the wild and single mutant strain DE17?yeaP,the number of bacteria that distribution in liver and spleen organs was significantly decreased(p<0.01),also the number of bacteria of double gene mutant strain DE17?luxS?yeaP that adhesion of DF-1 cell was significantly decreased(p<0.01),but its no significantly difference that comparing with DE17?luxS.The virulence test result showed that yeaP gene can promote the attenuation of virulence caused by the deletion of the luxS gene.The results of this study indicate that the LuxS/AI-2 quorum sensing system can regulate the biofilm formation of DE17 by regulating the c-di-GMP metabolic gene yeaP.The results of the promoter activity of the gene showed that p1-2 activity was the strongest.This study laid the foundation for further research on the regulation mechanism of LuxS/AI-2 quorum sensing system and c-di-GMP metabolic gene on biofilm formation in APEC.