| Bovine mastitis is mainly caused by microorganism infection.S.aureus is one of the most common causal agents.The ability of S.aureus to survie within a variety of mammalian cells protects it from the clearance by immune system,resulting in chronic infection.Therefore,further elucidation of S.aureus persistence mechanisms in the mammary gland is necessary to control the disease.Recent studies have shown that various pathogens modulate mitophagy to achieve immune escape.However,the effects of mitophagy in bovine mammary gland infected with S.aureus remain unclear.In this study,bovine mammary epithelial cell line(MAC-T)was used to establish the model of S.aureus intracellular infection.Mitophagy induced by S.aureus infection was investigated by flow cytometry,immunofluorescence,and Western blotting.This study will provide scientific theoretical basis for clinical prevention and treatment of mastitis caused by S.aureus.1.Study on the mitophagy induced by S.aureus in bovine mammary epithelial cellsThe MAC-T cells and S.aureus clinical isolate were used to establish intracellular infection model.Mitophagy induced by S.aureus infection were detected by flow cytometry,immunofluorescence,Western blotting and transmission electron microscopy.S.aureus infection resulted in a significant increase of ROS levels(p<0.01)and a significant loss in mitochondrial transmembrane potential(p<0.05 or p<0.01).Damaged mitochondrial structure was observed in S.aureus-infected MAC-T cells.S.aureus infection significantly enhanced the localization of mitochondria with autophagosome(p<0.01)and lysosome.Furthermore,damaged mitochondria were enclosed by autolysosomes in S.aureus-infected cells.The protein expression level of LC3II was significantly increased(p<0.01)and the protein expression of TOM20 and TIM23 were significantly decreased after S.aureus infection(p<0.05 or p<0.01).The levels of mtDNA were significantly reduced(p<0.05 or p<0.01)in infected cells.The decreased levels of TOM20 and TIM23 induced by S.aureus infection could be recovered by chloroquine treatment,indicating that S.aureus infection is able to induce lysosome-mediated degradation of mitochondria.These results demonstrate that S.aureus infection resulted in mitochonrrial damage and mitophagy in MAC-T cells.2.Study on the role of mitophagy in bovine mammary epithelial cells induced by S.aureus based on Pink1/Parkin pathwayTo explore the role of Pink1/Parkin pathway in S.aureus-induced mitophagy,cell models with silenced and overexpressed Pink1 were established by interfering RNA technology and lentivirus transfection strategy in MAC-T cells.The results showed that the protein expression of Pink1 was significantly upregulated(p<0.05),while parkin protein expression was not changed obviously.Western blot analysis of mitochondrial protein showed that the protein levels of Pink1 and Parkin were significantly increased in the mitochondrial fraction(p<0.05 or p<0.01).Furthermore,Pink1 silencing inhibited S.aureus-induced degradation of TOM20 and TIM23(p<0.05)as well as colocalization of mitochondria with autophagosome(p<0.01).Pinkl overexpression significantly enhanced S.aureus-induced degradation of TOM20 and TIM23(p<0.05 or p<0.01)as well as colocalization of mitochondria with autophagosome(p<0.01).The results above indicated that the Pink1/Parkin pathway played an important role in S.aureus-induced mitophagy in MAC-T cells.3.The effects of Pink1/Parkin-mediated mitophagy on the activation of S.aureus-induced NLRP3 inflammasome and NF-κB signaling pathwayThe activation of NLRP3 inflammasome and the phosphorylation levels of p65 and IκBα proteins were detected by Western blotting to explore the effects of Pink1/Parkin-mediated mitophagy on the inflammatory response induced by S.aureus in MAC-T cells.The results showed that the protein levels of NLRP3,Caspase-1 p20,and IL-1β p17 and the phosphorylation levels of p65 and IκBα proteins were significantly elevated after S.aureus infection(p<0.05 or p<0.01).Compared with cells infected with S.aureus alone,elevated protein amounts of NLRP3,Caspase-1 p20 and IL-1βp17 and enhanced phosphorylation of p65 and IκBα were observed after S.aureus infection and Mdivi-1 treatment(p<0.05 or p<0.01).Compared to the NC group,the NLRP3,Caspase-1 p20,and IL-1β p17 levels were significantly upregulated(p<0.05)and the phosphorylation levels of p65 and IκBα proteins were also increased(p<0.05 or p<0.01)in NC+S.aureus group,and more significant increases of above protein expressions and phosphorylation levels were observed after silencing Pinkl(p<0.05 or p<0.01).Compared to the NC+S.aureus group,Pinkl overexpression significantly downregulated the NLRP3,caspase-1 p20,and IL-1β p17 levels(p<0.05 or p<0.01)as well as the phosphorylation levels of p65 and IκBα proteins(p<0.01).Collectively,Pink1/Parkin-mediated mitophagy inhibited S.aureus-induced NLRP3 inflammasome and NF-κB signaling pathway activation.4.Study on the role of mtROS in inflammatory response of MAC-T cells induced by S.aureusTo investigate the role of mtROS in the inflammatory response induced by S.aureus,the changes of mtROS and the activation of NLRP3 inflammasone and NF-κB signaling pathway were detected by flow cytometry and Western blotting,respectively.The load of S.aureus in MAC-T cells was detected to investigate the effects of Pink1/Parkin-mediated mitophagy on S.aureus survival.The results showed that the production of mtROS was significantly increased after silencing Pink1(p<0.01),while the production of mtROS was significantly inhibited after Pinkl overexpression(p<0.01)in S.aureus-infected cells.Western blot analysis showed that silencing Pink1 significantly increased the protein levels of NLRP3,Caspase-1 p20,and IL-1β p17(p<0.05 or p<0.01)as well as the phosphorylation levels of p65 and IκBα proteins(p<0.05 orp<0.01)in S.aureus-infected cells compared with the NC+S.aureus group.Compared to the siPink1+S.aureus group,the protein levels of NLRP3,Caspase-1 p20,and IL-1β p17as well as the phosphorylation levels of p65 and IκBα proteins were significantly decreased in the siPink1+S.aureus+MitoTEMPO group(p<0.01).Compared to the control group,Mdivi-1 or silencing Pink1 significantly decreased S.aureus load in MAC-T cells(p<0.01),Pink1 overexpression significantly increased the S.aureus load in MAC-T cells(p<0.01).The results demonstrate that Pink1/Parkinmediated mitophagy inhibited S.aureus-induced NLRP3 inflammasome and NF-κB signaling pathway activation via reducing the production of mtROS.Furthermore,Pink1/Parkin-mediated mitophagy promoted S.aureus survival in MAC-T cells.In conclusion,S.aureus infection resulted in mitochondrial damage and induced Pink1/Parkin-mediated mitophagy in MAC-T cells.Pink1/Parkin-mediated mitophagy inhibited S.aureus-induced NLRP3 inflammasome and NF-κB signaling pathway activation by decreasing the production of mtROS.Furthermore,S.aureus may hijack mitophagy process for its survival in MAC-T cells. |
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