| Staphylococcus aureus(S.aureus)is the main pathogen of mastitis in dairy cows,which can internalize cells.Intracellular S.aureus can colonize cells by adapting to the internal environment of host cells.Xenophagy is a form of selective autophagy to capture intracellular bacteria within the autophagosomes,for degradation in the lysosome.Bacterial invasion and host cells’ resistance to infection through xenophagy are processes in which pathogens interact with host cells,and there may be a complex regulatory network in this process,but the mechanism is not fully understood.Bovine mammary epithelial cells(BMECs)are the main cells of the dairy cow mammary gland.BMECs are commonly used in the study of bovine mastitis.In this study,BMECs were used as the cell model to explore the changes of m RNA expression profile and protein expression profile after bacterial infection through transcriptome and proteome analysis,to screen out the key molecules of host cells against infection and clarify the molecular mechanism of xenophagy induced by S.aureus.These results provided a new insight for the control of S.aureus-induced bovine mastitis.An intracellular S.aureus infection model of BMECs was established,and the differentially expressed genes and proteins associated with S.aureus invasion were screened out by transcriptome analysis and proteomic reanalysis.The functions of ITGAV,ACTR2,ACTR3 and CI-MPR were verified to explore the role of them in bacterial infection;Further,the xenophagy induced by S.aureus was detected by TEM,immunofluorescence staining and other methods.The functions of CI-MPR and Clathrin in xenophagy were verified by means of post-transcriptional silencing and inhibitor treatment.Furthermore,the activities of PI3K/Akt signaling pathway and JNK/c-Jun signaling pathway were detected after S.aureus treatment,to explore the regulatory effects of JNK/c-Jun signaling pathway on the expression of CI-MPR and xenophagy.Results showed that:1)The bacteria were evaluated intracellularly and extracellularly by bacterial colony count,S.aureus was observed in whole-cell lysates,whereas none was found in the culture medium.Further,intracellularly stained S.aureus was seen under an LSCM,and S.aureus was found free in the cytoplasm of BMECs by TEM.Transcriptome analysis of S.aureus-infected BMECs at 2 h and 8 h was performed,respectively,and three differentially expressed genes related to bacterial invasion were screened,which were ITGAV,ACTR2 and ACTR3.2)Proteome data of BMECs infected with S.aureus for 2 h and control group were reanalyzed.It was found that CI-MPR was annotated into lysosome,phagosome and endocytosis entries in KEGG database,suggesting that CI-MPR might be involved in the defense against S.aureus infection.Western blot analysis showed that the expression level of CI-MPR was increased in S.aureus-infected cells.3)ITGAV post-transcriptional silencing and Integrin inhibitor RGD treatment significantly reduced the intracellular number of S.aureus,suggesting that Integrin mediated S.aureus invasion of BMECs.The number of S.aureus in BMECs was significantly reduced by ACTR2 and ACTR3 post-transcriptional silencing,suggesting that ACTR2 and ACTR3 mediate S.aureus invasion of BMECs.4)The number of S.aureus in BMECs was significantly increased after CI-MPR posttranscriptional silencing and CI-MPR antibody blocking;The number of S.aureus in BMECs was significantly decreased after treatment with CI-MPR agonist D-M6 P.The number of S.aureus invasion into BMECs was significantly increased after Clathrin inhibitor Pitstop 2 treatment.Bioinformatics prediction results and coimmunoprecipitation results elucidate an interaction between CI-MPR and Clathrin.These results indicate that CI-MPR couples Clathrin to resist S.aureus invasion of BMECs.5)After S.aureus invaded BMECs,autolysosome containing S.aureus was observed in BMECs by TEM.The co-localization of S.aureus with LC3 was observed by LSCM,the number of MDC positive structures increased.Western blot results showed that the expression levels of p62 and LC3 were increased.After treated with autophagy inhibitor HCQ,xenophagy was blocked.6)CI-MPR post-transcriptional silencing reduced xenophagy induced by S.aureus invasion;After D-M6 P treatment,xenophagy enhanced.Clathrin inhibitor Pitstop 2treatment attenuated xenophagy induced by S.aureus invasion.These results indicate that both CI-MPR and clathrin mediate S.aureus-induced xenophagy.7)S.aureus invasion of BMECs activates PI3K/Akt signaling pathway and JNK signaling pathway.CI-MPR mediated the activation of PI3K/Akt and JNK signaling pathways induced by S.aureus invasion of BMECs.PI3 K inhibitor LY294002 and JNK inhibitor SP600125 treatment proved that PI3K/Akt signaling pathway has a regulatory effect on JNK signaling pathway.8)Bioinformatics predicted that c-Jun was a transcription factor of CI-MPR.JNK/cJun signaling pathway was activated and the expression of CI-MPR increased after S.aureus invasion.After JNK inhibitor SP600125 treatment,the activity of JNK/c-Jun was inhibited,the expression level of CI-MPR was decreased,and xenophagy was weakened.It is suggested that JNK/c-Jun signaling pathway regulates CI-MPR gene expression and xenophagy.In conclusion,S.aureus invades BMECs through Integrin and ARP2/3 complex and induces xenophagy.CI-MPR and Clathrin work together to resist S.aureus invasion and mediate xenophagy.Meanwhile,bacterial invasion led to activation of PI3K/Akt signaling pathway and JNK/c-Jun signaling pathway in host cells,which promoted the expression of CI-MPR.CI-MPR/PI3K-Akt coupled with JNK/c-Jun to regulates xenophagy and resist bacterial infection.These results demonstrated a new mechanism by which S.aureus invades BMECs and BMECs resist S.aureus infection,providing a theoretical basis for the prevention and treatment of bovine mastitis. |
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