The Effect Of Staphylococcus Aureus Infection On The Expression Of Key Enztmes In Amino Acids Metabolism In Human Mammary Epithelial Cell

Staphylococcus aureus(S.aureus)is one of the major pathogens that cause infections in mammals,and it may cause mastitis through infecting into mammary gland.Mastitis is harmful to people and livestock,it may also affect the quantity and quality of dairy products.Milk protein is an important composition of dairy products.The kinds and content of amino acid in mammary epithelial cells directly affect the synthesis of milk protein,and amino acid metabolism is an important factor that influences the synthesis of milk protein.S.aureus infection affects the synthesis and secretion of milk protein in mammary epithelial cells;however,the influence on amino acids metabolism and its mechanism is still unclear.Human mammary epithelial cell(MCF-10A)was used as model in this study,S.aureus(ATCC27543)was used to infect cells,and the expression of six key enzymes in amino acid metabolism and β-casin were detected.Such key enzymes include Aspartate aminotransferase(AST),Glutathione S-transferase(GST),Glutamate Dehydrogenase(GDH),Oxoglutarate dehydrogenase(OGDH),Ornithine Decarboxylase(ODC)and Glutamic acid decarboxylase(GAD),which are involved in transamination,deamination and decarboxylation processes,respectively.Firstly,MCF-10A cells were infected with S.aureus.The expression of key enzymes in amino acids metabolism and β-casin were detected by qPCR method,the synthesis and secretion of related gene products in and out of cells were measured by ELISA method,the activity of mTORC1 signaling pathway was tested by Western blot.And then,PGN was used to stimulate MCF-10A cells,in the same way,the expression and secretion of key enzymes in amino acids metabolism and β-casin were detected by qPCR and ELIS A methods,the activity of mTORC1 signaling pathway and several transcription factors were tested by Western blot.Finally,on the basis of the first two sections of experiments,we explored the regulation of mTORC1 signaling on the activity of transcription factors and the expression of key enzymes in amino acids metabolism.After inhibiting the mTORCl signaling through rapamycin,a specific chemical inhibitor of mTORC1,and RNAi method,combined with S.aureus infection or not,the expression of key enzymes,the synthesis and secretion of P-casin were measured at mRNA level and protein level,respectively;and the activity of several transcription factors that may related to amino acids metabolism were also detected.The results show that:(1)S.aureus can infect into MCF-10A,the content of cellular key enzymes in amino acids metabolism shows a tendency of firstly increased and then slightly declined with the infection time.After infection for 8 h,the synthesis and secretion of β-casin significantly declined(p<0.001).Meanwhile,the activity of mTOR signaling also shows a tendency of firstly increased and then declined;(2)PGN stimulation induced the expression of key enzymes in amino acids metabolism,and the cellular content of which were significantly increased(p<0.001),while the synthesis and secretion of β-casin have no significant changes(p>0.05).Additionally,the activity of mTOR signaling and several transcription factors were increased;(3)Rapamycin can remarkably inhibit the induced expression of key enzymes in amino acids metabolism by S.aureus infection and PGN stimulation(p<0.01),which caused significant decrease in the content of cellular key enzymes in amino acids metabolism(p<0.01),the synthesis and secretion of β-casin were also inhibited(p<0.01),the activity of several transcription factors and mTOR signaling declined at different levels;(4)RNAi method was used to interference the key component of mTORC1,raptor,so as to inhibit mTORC1 signaling,the results were consistent with the above rapamycin-treated data,verifying that mTORC1 signaling has regulatory effect on the expression of key enzymes in amino acids metabolism and the synthesis and secretion of and β-casin.In summary,this experiment demonstrated that S.aureus can infect into MCF-10A,affectig the key enzymes in amino acids metabolism and the synthesis and secretion of P-casin.The.activity of mTOR signaling shows a tendency of firstly increased and then declined with the infection time,the content of cellular key enzymes in amino acids metabolism also shows a tendency of firstly increased and then slightly declined with the infection time,in addition,the synthesis and secretion of β-casin are decreased with the infection time.Peptidoglycan of S.aureus(PGN)can induce the expression of six key enzymes in amino acids metabolism,activate mTORC1 signaling and related transcription factors.mTORC1 signaling mediates S.aureus infection and PGN stimulation,which may regulate the expression of GST,AST,GDH,OGDH,ODC and GAD and the synthesis and secretion of β-casin through influencing the activity of transcription factors NF-κB,STAT1,STAT3,SREBP1 and PPARγ.Our study provides the basic data with a further exploration on the regulation of mTORC1 signaling on amino acids metabolism and its mechanism.