Staphylococcus Aureus Invades Bovine Mammary Epithelial Cells And Influences Milk Fat Synthesis

Milk fat is one of the important nutrients in milk,and an important indicator of dairy products quality and feed efficiency,and therefore the milk fat synthesis and regulation mechanism in dairy cows becomes a great concern in ruminant nutrition.Dairy cow mastitis is the inflammation of the mammary gland accompanied by physiological and chemical changes of breast,and has the highest incidence in dairy farming,with the fastest prevalence and the most significant losses.One of the main causes of dairy cow mastitis is pathogenic microorganisms,and Staphylococcus aureus is the most common pathogenic bacteria causing mastitis.At present,the effect on milk fat synthesis of S.aureus invading bovine mammary gland epithelial cells and its regulatory mechanism are still unclear.In this academic dissertation,the bovine mammary gland epithelial cells(BMECs)were obtained by primary culture and then BMECs were separated and purified.A high definition laser confocal microscope was used to observe thecytoskeleton rearrangement caused by S.aureus invasion.Colony counting method was applied to determine the number of bacteria invaded in cells.ELISA was performed to measure the content of triglyceride(TAG),palmitic acid(PA)and docosahexaenoic acid(DHA)in the cell culture medium,and mass spectrometry(MS)to determine the intracellular concentration of 37 types of fatty acid.Western Blot was carried out to detect the activity of mTORC1 signaling pathway and expression of lipogenesis related transcription factors and key enzymes.Yeast two-hybrid and Co-immunoprecipitation(Co-IP)assays were performed to find the interaction between proteins,and to provide data for understanding the molecular mechanism of the disorders in milk fat synthesis caused by S.aureus invasion.The results showed that 1)the cytoskeleton rearrangement was observed under the laser confocal microscope when the BMECs invaded by S.aureus for 2 h;2)the number of bacteria in the cells increased with the extending of invasion time;3)BMECs morphological changes were observed under the optical microscope,accompanied by a decrease in the cell number with the increasing of invasion time and bacteria number;4)the content TAG,PA and DHA in cell medium showed significantly decreased when S.aureus invasion for 4 h~16 h(p<0.05).The activity of mTORC1 signaling pathways and the expressions of PPAR?,SREBP1,Lipin1,ACC and FAS showed a trend for firstly increased and then decreased in the period of4 h~16 h with S.aureus invasion;5)the intracellular concentrations of total fatty acid,saturated fatty acid and unsaturated fatty acid were significantly decreased when bacteria invasion for 8 h(p<0.05),suggesting that the bacteria invasion inhibited thesynthesis of fatty acids in the cells;6)confirmed the direct interaction of Lipin1 with PPAR? was identified by 3 assays including MS,Co-IP and yeast two-hybrid,and there are 372 binding proteins which shared by them were determined by MS,among which 108 proteins showed an interaction network according to bioinformatics prediction;7)Q95KV1,which is an endogenous inhibitor of IKK?,was noticed by us based,on manual selection,and then IKK? and I?B? were detected in the Co-IP production by western blot,suggesting a possible relevence between lipogenesis related proteins Lipin1 and PPAR? and inflammatory-related proteins.The data provide the basis for studying the interaction between inflammation and lipid metabolism in the futrue.In conclusion,S.aureus invades BMECs can lead to cytoskeleton rearrangement,cell deformation and lysis,and inhibits the synthesis of milk fat.The activity of mTORC1 signaling pathway and the expressions of the lipogensis related transcription factors and key enzymes were increased firstly and then decreased in period of 4 h~16 h with S.aureus invasion.Lipin1 has a direct interaction with PPAR? and is associated with inflammatory-related functional proteins--IKK? and I?B?,providing a foundation for the study on relationship of lipid metabolism and inflammatory responses.Through a mastitis cell model,this academic dissertation demonstrates that S.aureus invasion has inhibitive effects on milk fat synthesis and a possible molecular mechanism was discussed,which is of great significance in understanding the pathogenesis of dairy cow mastitis,improving the quality of dairy products,and enhancing the core competition of the dairy industry.