Molecular Mechanism Of Enterococcus Faecalis From Cow Mastitis Escaping Complete Phagocytosis Of Cow Macrophages Based On The Core Virulence Factor CylA Gene

This experiment was to study Enterococcus faecalis from bovine mastitis β Molecular mechanism of hemolysin cylA gene on phagocytic function and immune regulation of bovine macrophages.The wild type strains and cylA gene deletion mutants of Enterococcus faecalis isolated clinically from bovine mastitis were used to infect bovine macrophages.The cell activity was detected by MTT assay,and the following tests were performed under the condition that the cell activity was not affected.The total m RNA of infected macrophages were extracted and the cytokines IL-4,IL-8,IL-10,IL-12 and TNF-α were detected,And the transcriptional level of NOS2,by RT-qPCR.Detection of cytokines IL-4,IL-8,IL-10,IL-12 and TNF-α by ELISA.The expression of Western blot was used to clarify the molecular mechanism of its regulation of this signal pathway,which could detect the phosphorylation level of cylA gene of Enterococcus faecalis to p38 MAPK signal pathway.After incubating macrophages with p38 MAPK signal pathway inhibitor before the infection experiment,the infection experiment was carried out.The cytokines IL-4,IL-8,IL-10,IL-12 and TNF-α were detected by ELISA.To study the effect of p38 MAPK signal pathway inhibitor on cytokine expression.After RT-qPCR detection,it was found that compared with the cylA gene knockout strain group,the cylA positive wild type strains of Enterococcus faecalis in IL-10 and TNF-α The level of transcription was increased,while the levels of IL-4,IL-8,IL-12 and NOS2 were decreased,which were statistically significant.The ELISA results were basically consistent with RT-qPCR,in which TNF-α The expression level of the gene was improved,but it was not statistically significant.Western blot was used to detect the phosphorylation level of p38 MAPK.Compared with the wild type group of Enterococcus faecalis with cylA positive,the expression of p-p38 in the cylA gene knockout group decreased significantly.After adding p38 MAPK inhibitor,ELISA results showed that compared with the group without p38 MAPK inhibitor,IL-10 and TNF-α in the group with p38 MAPK inhibitor The expression level of IL-4,IL-8 and IL-12 was increased,and the expression level was statistically significant.According to the above results,p38 MAPK signal pathway was Enterococcus faecalisβ The hemolysin cylA gene plays an important role in the immune regulation mechanism,which can provide a new target for the treatment and prevention and control of cow mastitis.The inactivation of its virulence and the identification and blocking of p38 MAPK signal pathway can effectively prevent and control Enterococcus faecalis,and provide a new solution to the problem of milk safety caused by Enterococcus faecalis.