Study On The Mechanism Of Streptococcus Agalactiae Invasion Of Bovine Mammary Epithelial Cells

Mastitis is a common disease in dairy cows Streptococus agalactiae is one of the pathogenic bacteria causing mastitis.In addition to colonizing in mammary tissue,S.agalactiae can invade bovine mammary epithelial cells(BMECs)through adhesion.The host cells resist bacterial invasion through gene expression and programmed cell death.At present,the mechanism by which S.agalactiae invades BMECs and host cells against bacterial invasion is unclear.Pyroptosis is lytic,and inflammatory programmed-cell death,which is mainly induced by intracellular bacteria.It is a new mechanism of organism against pathogen infection discovered in recent years,and whether S.agalactiae can induce pyroptosis in BMECs has not been reported.In the present study,primary BMECs(pBMECs)were isolated and cultured by the adherent culture of enzymatic digestion of cow mammary tissue using collagenase,and pure pBMECs were used as a model to explore the mechanism of invasion in pBMECs by S.agalactiae and whether it can induce pyroptosis.The spread plate method and laser scanning confocal microscopy(LSCM)were used to determine amount of intracellular S.agalactiae;the comparative proteomics was used to analyze and find differentially expressed proteins between S.agalactiae invasion cells and control cells,and then a few differentially expressed proteins were verified by western blot;LAMB2 silencing cells,LAMB2 blocked cells,integrin inhibited cell by inhibitor(RGD),ITGAV silencing cells,FAK inhibited cells by a small molecule inhibitor(TAE226),FAK silencing cells,Crk silencing cells,Crk over-expressed cells,Vps25 silencing cells,and Vps25 over-expressed cells were invaded by S.agalactiae,respectively.The intracellular bacteria were counted by the spread plate method and qPCR;the RGD treated cells,TAE226 treated cells,FAK silencing cells,Crk silencing cells,and Vps25 silencing cells were invaded by S.agalactiae,and then the expression of proteins in the treated cells and control cells was detected by Western blot;the pyroptosis induced by S.agalactiae was examined by Western blot,ELISA and scanning electron microscopy.The results showed that: 1)S.agalactiae could successfully invade into pBMECs at different MOIs after two hours;2)169 differentially expressed proteins were screened out in S.agalactiae invaded pBMECs,compared with control,and 37 differentially expressed proteins were screened by GO and KEGG analysis,including 16 up-regulated proteins and 21 down-regulated proteins;3)The protein Crk,Vps25,and RhoA were selected as the target proteins and verified by Western blot based on the purpose of this study.The results were consistent with those of proteomics.In the S.agalactiae invasion pBMECs,the protein Crk was up-regulated,while Vps25 and RhoA were down-regulated;4)The number of S.agalactiae invaded into pBMECs was significantly inhibited by LAMB2 silencing or LAMB2 antibody blocking(p<0.05).5)The result indicated that LAMB2 mediated the invasion of S.agalactiae;The number of S.agalactiae invaded into pBMECs was significantly inhibited by RGD or ITGAV silencing(p<0.05).6)The result indicated that integrin mediated the invasion of S.agalactiae;The number of S.agalactiae invaded into pBMECs was significantly decreased by TAE226 or FAK silencing(p < 0.05).7)The result indicated that FAK promotes the invasion of S.agalactiae;The number of S.agalactiae invaded into pBMECs was decreased by Crk over-expression(p<0.05),or increased by Crk silencing(p<0.05),respectively.8)The result indicated that Crk prevents the invasion of S.agalactiae;The number of S.agalactiae invaded into pBMECs was decreased by Vps25 over-expression(p<0.05),or increased by Vps25 silencing(p<0.05),respectively.The result indicated that Vps25 prevents the invasion of S.agalactiae;9)The invasion of S.agalactiae promotes the level of FAK phosphorylation,and inhibites Crk phosphorylation and the expression of Vps25 and RhoA.These results indicated that the invasion of S.agalactiae inhibits Crk activity by activating FAK,and then inhibits Vps25 expression to weaken the ability of cells against the bacteria invasion.Meanwhile,the invasion of S.agalactiae inhibits RhoA expression,suggesting that it could promote cytoskeletal rearrangement and facilitate bacteria internalization;10)Crk interacts with Vps25 and they may jointly defend against the invasion of S.agalactiae;11)The Caspase-1 was activated,and the secretion of IL-1? and IL-18 was significantly increased,accompanied with the forming of the small holes in the cell membrane,after S.agalactiae invaded pBMECs at MOI60 for 24 h.These results indicated that pyroptosis was induced by invasion of S.agalactiae.In conclusion,S.agalactiae can attach to the cell membrane by binding to the extracellular matrix laminin(LAMB2),and invade pBMECs through the transmembrane protein integrin.The invasion of S.agalactiae promotes the activity of FAK,and inhibits the activity of Crk and the expression of Vps25 and RhoA,resulting in cytoskeletal rearrangement,facilitating bacteria internalization.In this process,Crk and Vps25 play a critical role of in preventing the invasion of S.agalactiae into pBMECs,and the activited FAK may be beneficial to the invasion of S.agalactiae.The intracellular S.agalactiae induced pyroptosis.The results of our study will provide basic data for prevention and therapic strategy of dairy cow mastitis.