The Role Of C-di-GMP Metabolism Genes And STING-IRF3 Signaling Pathway In Bovine Mastitis Escherichia Coli Infection

Escherichia coli is one of the most common udder pathogens associated with acute clinical mastitis,however,its pathogenic mechanism still needs to be elucidate.Cyclic diGMP is a ubiquitous second messenger that regulates polysaccharide synthesis,motility,and virulence in diverse bacterial species.It plays an important role in the process of infecting host via regulating STING-IRF3 signaling pathway of the host cell.There are 8 conserved genes in E.coli which are c-di-GMP synthesis and degradation genes（yeaJ,yegE,yneF,yciR,yfgF,ydiV,yeaI and csrD）.These genes participate in regulating biofilm formation,adhesion to and invasion of host cells.However,the regulatory functions of metabolism gene are largely unknown in mastitis E.coli.Therefore,the present study used E.coli NJ17 strain as the research object and constructs the above-mentioned 8 genes mutants to clarify the role of c-di-GMP metabolism genes and STING-IRF3 signaling pathway in dairy cows mastitis caused by E.coli.Our study will provide a reference for the prevention and control of E.coli mastitis.The research results are as follows:1.Construction of 8 c-di-GMP metabolism gene mutants of E.coli NJ17 and analysis of their biological characteristicsIn this study,E.coli NJ17 strain was used as the research object.PCR result show that 28 c-di-GMP metabolism genes are existed in E.coli NJ17 and 8 c-di-GMP metabolism gene mutants of dairy cow mastitis-derived E.coli NJ17 were successfully constructed with λ-Red homologous recombination technology（NJ17-ΔyeaJ,NJ17-ΔyegE,NJ17-ΔyneF,NJ17ΔyciR,NJ17-ΔyfgF,NJ17-ΔydiV,NJ17-ΔyeaI and NJ17-ΔcsrD）.In order to investigate the effect of c-di-GMP metabolism gene on the biological characteristics of dairy cows mastitisderived E.coli NJ17,growth curve,biofilm formation,rdar morphology,motility and drug resistance of the wild strain NJ17 and its eight c-di-GMP metabolism-related genes mutants are determined.The results show that the deletion of 8 c-di-GMP metabolism genes cannot affect the growth characteristics and drug resistance of E.coli NJ17,but it reduced the biofilm formation ability.The partly deletion of the c-di-GMP metabolism genes（yneF,ydiV,csrD,yeal,and yeaJ）altered the motility of the E.coli NJ17.This study lays the foundation for further research on the regulatory function of E.coli NJ17 c-di-GMP metabolism genes.In order to explore the regulation of 8 c-di-GMP metabolism genes on important target genes of E.coli NJ17,Real-time PCR test results showed that the deletion of ydiV and yciR significantly reduced transcription level of genes（wzy and lptA）related to the synthesis and transport of LPS.The deletion of yeaJ,yneF and csrD genes significantly increased the transcription level of ompA（outer membrane protein A）and eaeA（type Ⅲ secretion system related gene）.This study lays the foundation for further research on the regulatory function of E.coli NJ17 c-di-GMP metabolism genes.2.Study on the role of c-di-GMP metabolism genes of E.coli NJ17 in the infection of EpH₄Ev and RAW264.7In order to investigate the role of 8 c-di-GMP metabolism genes of E.coli NJ17 isolated from bovine mastitis in infection of host cells.Mouse mammary epithelial cells（EpH₄-Ev）and mouse macrophage（RAW264.7）cell lines were selected to compare the differences between wild strain NJ17 and its 8 deleted strains in interaction with host cells.Compared to wild strains,NJ17-ΔydiV significantly improved the adhesion and invasion to EpH₄-Ev and RAW264.7 cells.The IFN-(3 level and LDH enzyme activity in cell supernatant of RAW264.7 as well as the IL-6 content in EpH₄-Ev cell supernatant signifcantly improved.At 6-12 h,NJ17-ΔydiV also significantly improve the survival rate in RAW264.7（P<0.05）.Compared to wild strains,NJ17-ΔyeaJ gene significantly reduced the adhesion and invasion to EpH₄Ev and RAW264.7 cells.However,the IFN-β and IL-6 levels in the cell supernatant of EpH₄Ev and RAW264.7 cells are significantly increased.At 6-12 h,NJ17-ΔyeaJ significantly reduce the survival rate in RAW264.7（P<0.05）;NJ17-ΔyegE only significantly increased adhesion to EpH₄-Ev cells and LDH enzyme activity in the supernatant.NJ17-ΔcsrD also significantly decreased the LDH enzyme activity in the supernatant of RAW264.7 cells and the survival rate in RAW264.7 cells.Other gene deletion had no significant effect on the interaction of E.coli NJ17 with EpH₄-Ev and RAW264.7 cells.The results of this study show that yeaJ and ydiv genes play an important role in host cells infected by E.coli NJ17 isolated form bovine mastitis.3.The role of STING-IRF3 signaling pathway in the infection of EpH₄-Ev and RAW264.7 by E.coli NJ17In this study,yeaJ gene is used as research object which is screened and identified for playing an important regulatory role in NJ17 infection of EpH₄-Ev and RAW264.7 cells.Furthermore,the role of c-di-GMP,yeaJ gene（encoding c-di-GMP synthase）and STINGIRF3 signaling pathway in the NJ17 infection of EpH₄-Ev and RAW264.7 cells were analyzed.In this study,the effects of different concentrations of c-di-GMP on STING-IRF3 signaling pathway in EpH₄-Ev cells were studied by exogenous addition or liposome transfection.Western Blot detection results showed that when 4 μg/mL c-di-GMP was adding to EpH₄-Ev,the protein expression of TBK1,STING and IL-6 decreased significantly compared with the control group（P<0.05）.When 4 μg/mL c-di-GMP was transfected with liposome,the protein expression of TBK1,STING and IL-6 in EpH₄-Ev was significantly increased（P<0.05）.In this study,immunofluorescence,fluorescence quantification and western blot detection were used to study the role of yeaJ and STINGIRF3 signaling pathway during NJ17 infection of EpH₄-Ev and RAW264.7 cells.During NJ17 infection of EpH₄-Ev and RAW264.7,it could promote the binding ability of STING and TBK1 and activates STING-IRF3 signaling pathway.Compared with the wild strain,NJ17-ΔyeaJ significantly increased the transcription level of STING and IRF3 and enhanced the binding ability of STING and TBK1.However,compared with wild strain,NJ17-ΔyeaJ significantly decreased the transcription and protein expression of STING and IRF3 and the binding ability of STING and TBK1.These results showed that the deletion of yeaJ gene in bovine mastitis Escherichia coli promotes the activation of STING-IRF3 signaling pathway in EpH₄-Ev.However,it inhibit the activation of STING-IRF3 signalling pathway in RAW264.7.The results of this study showed that the c-di-GMP metabolism gene in bovine mastitis E.coli played significant role in its biological characteristics.STING-IRF3 signaling pathway and c-di-GMP metabolism genes played an important role in mastitis caused by E.coli.This study provides a theoretical base to explore the role of bacterial c-di-GMP and host STINGIRF3 signaling pathway in dairy mastatic cows.