Streptococcus agalactiae Affects Milk Protein Synthesis in Bovine Mammary Epithelial Cells by Inhibiting Tyrosine Kinase/Signal Transduction and Transcriptional Activators and Mammalian Target of Rapamycin Signaling Pathways
YANG Delian1, TONG Jinjin1, SUN Mingwei1, ZHANG Jie1, ZHANG Hua1, XIONG Benhai2, JIANG Linshu1
1. Key Laboratory for Dairy Cow Nutrition, College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China;
2. Institute of Animal Science and Veterinary, Chinese Academy of Agricultural Science, Beijing 100193, China
Abstract：The objective of this study was to explore the mechanism of the effects of Streptococcus agalactiae (GBS) on milk protein synthesis in bovine mammary epithelial cells (BMECs). Three different concentrations of GBS (MOI=100, 50 and 10) were used to infect BMECs for 1, 2, 4, 6, 8, 12, 18, and 24 h, and each time point was set up corresponding blank control. Then,the effects of GBS on the activity, morphology and apoptosis of BMECs were determined using lactate dehydrogenase (LDH) kit, scanning electron microscopy and flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein expression of related genes of β-casein and lactoprotein synthesis. The results showed as follows:1) the toxicity of GBS to BMECs was time-and dose-dependent. With the prolongation of infection time and the increase of bacterial concentration, LDH release amount was significantly or extremely significantly higher than that of the control group (P<0.05 or P<0.01). The cell morphology changed significantly after GBS infection. At 6 h of infection, the cell structure was broken and the cell morphology was severely damaged at 8 h of infection. Moreover, the apoptosis of BMECs was significant at 2 h (P<0.05) and extremely significant at 6 h of infection (P<0.01). 2) When the infecting time lasted for 6 h, GBS resulted that the mRNA expression levels of β-casein and the genes regulating lactoprotein expression like tyrosine kinase 2 (JAK2), signal transduction and transcription activator 5a (STAT5a), mammalian target of rapamycin (mTOR), serine/threonine protein kinase 1 (AKT1), ribosomal protein S6 (sRPS6), prolactin receptor (PRLR) and est domain transcription factor 5 (ELF5) in BMECs, were extremely significant down-regulated (P<0.01), while the mRNA expression level of negative regulatory gene eukaryotic translation initiation factor 4E binding protein 1 (EIF4E-BP1) was remarkably up-regulated (P<0.01). The protein expression levels of β-casein, STAT5a, p-STAT5a, mTOR, p-mTOR, and AKT1 in BMECs were dramatically down-regulated by GBS (P<0.01), while P-AKT1 protein expression was completely inhibited. At 8 h of infection, GBS completely inhibited the protein expression of β-casein, STAT5a, p-STAT5a. It can be concluded that GBS can damage the morphological structure of BMECs, promote apoptosis and decrease cell viability, at last having an adverse impact on the normal cell physiological functions. In addition, GBS inhibits the lactoprotein synthesis in BMECs mainly by inhibiting the function of the JAK/STAT and mTOR signaling pathways.
PECKA-KIELB E,VASIL M,ZACHWIEJA A,et al.An effect of mammary gland infection caused by Streptococcus uberis on composition and physicochemical changes of cows' milk[J].Polish Journal of Veterinary Sciences,2016,19(1):49-55.
[14]
MOUSSAOUI F,MICHELUTTI I,LE ROUX Y,et al.Mechanisms involved in milk endogenous proteolysis induced by a lipopolysaccharide experimental mastitis[J].Journal of Dairy Science,2002,85(10):2562-2570.
[19]
RAINARD P,RIOLLET C.Innate immunity of the bovine mammary gland[J].Veterinary Research,2006,37(3):369-400.
[29]
GONÇALVES J L,KAMPHUIS C,MARTINS C M M R,et al.Bovine subclinical mastitis reduces milk yield and economic return[J].Livestock Science,2018,210:25-32.
[39]
HELTEMES-HARRIS L M,FARRAR M A.Constitutively active STAT5 constructs[J].Methods in Molecular Biology,2013,967:225-232.
[15]
ZHAO X,LACASSE P.Mammary tissue damage during bovine mastitis:causes and control[J].Journal of Animal Science,2008,86(13 Suppl):57-65.
[17]
CHEN X Y,DEGO O K,ALMEIDA R A,et al.Deletion of sua gene reduces the ability of Streptococcus uberis to adhere to and internalize into bovine mammary epithelial cells[J].Veterinary Microbiology,2011,147(3/4):426-434.
CHEN W,LIU Y X,ZHANG L M,et al.Nocardia cyriacigeogica from bovine mastitis induced in vitro apoptosis of bovine mammary epithelial cells via activation of mitochondrial-caspase pathway[J].Frontiers in Cellular and Infection Microbiology,2017,7:194.
[22]
CHEN W,LIU Y X,ZHANG L M,et al.Nocardia cyriacigeogica from bovine mastitis induced in vitro apoptosis of bovine mammary epithelial cells via activation of mitochondrial-caspase pathway[J].Frontiers in Cellular and Infection Microbiology,2017,7:194.
[28]
FLEMINGER G,RAGONES H,MERIN U,et al.Low molecular mass peptides generated by hydrolysis of casein impair rennet coagulation of milk[J].International Dairy Journal,2013,30(2):74-78.
[11]
BI Y L,WANG Y J,QIN Y,et al.Prevalence of bovine mastitis pathogens in bulk tank milk in China[J].PLoS One,2016,11(5):e0155621.
[9]
ZHANG M C,ZHAO S G,WANG S S,et al.D-glucose and amino acid deficiency inhibits casein synthesis through JAK2/STAT5 and AMPK/mTOR signaling pathways in mammary epithelial cells of dairy cows[J].Journal of Dairy Science,2018,101(2):1737-1746.
[12]
JØRGENSEN H J,NORDSTOGA A B,SVILAND S,et al.Streptococcus agalactiae in the environment of bovine dairy herds-rewriting the textbooks?[J].Veterinary Microbiology,2016,184:64-72.
[24]
KLING D E,TSVANG I,MURPHY M P,et al.Group B Streptococcus induces a caspase-dependent apoptosis in fetal rat lung interstitium[J].Microbial Pathogenesis,2013,61/62:1-10.
[23]
SHAHID M,GAO J,ZHOU Y N,et al.Prototheca zopfii isolated from bovine mastitis induced oxidative stress and apoptosis in bovine mammary epithelial cells[J].Oncotarget,2017,8(19):31938-31947.
[1]
嵇庆刚.牛奶中乳蛋白含量的影响因素及提高措施[J].现代畜牧科技,2016(11):31.
[16]
AKERS R M,NICKERSON S C.Mastitis and its impact on structure and function in the ruminant mammary gland[J].Journal of Mammary Gland Biology and Neoplasia,2011,16(4):275-289.
TIAN Q,WANG H R,WANG M Z,et al.Lactogenic hormones regulate mammary protein synthesis in bovine mammary epithelial cells via the mTOR and JAK-STAT signal pathways[J].Animal Production Science,2015,56(11):1803-1809.
GAO H N,HAN H U,ZHENG N,et al.Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway[J].Journal of Zhejiang University-SciEnce B,2015,16(6):560-572.
DAI W T,WHITE R R,LIU J X,et al.Seryl-tRNA synthetase-mediated essential amino acids regulate β-casein synthesis via cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells[J].Journal of Dairy Science,2018,101(11):10456-10468.
[6]
FILEP R,AKERS R M.Casein secretion and cytological differentiation in mammary tissue from bulls of high or low genetic merit[J].Journal of Dairy Science,2000,83(10):2261-2268.
[10]
LI M,ZHAO D,MA G,et al.Upregulation of ATBF1 by progesterone-PR signaling and its functional implication in mammary epithelial cells[J].Biochemical and Biophysical Research Communications,2013,430(1):358-363.
[18]
BOUCHARD D S,RAULT L,BERKOVA N,et al.Inhibition of Staphylococcus aureus invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei[J].Applied and Environmental Microbiology,2013,79(3):877-885.
[25]
DA COSTA A F E,MORAES J A,DE OLIVEIRA J S S,et al.Reactive oxygen species involved in apoptosis induction of human respiratory epithelial (A549) cells by Streptococcus agalactiae[J].Microbiology,2016,162(1):94-99.
[26]
EMANEINI M,KHORAMIAN B,JABALAMELI F,et al.Comparison of virulence factors and capsular types of Streptococcus agalactiae isolated from human and bovine infections[J].Microbial Pathogenesis,2016,91:1-4.
[27]
JAIN B,TEWARI A,BHANDARI B B,et al.Antibiotic resistance and virulence genes in Streptococcus agalactiae isolated from cases of bovine subclinical mastitis[J].Veterinarski Arhiv,2012,82(5):423-432.
[30]
FREEMAN M E,KANYICSKA B,LERANT A,et al.Prolactin:structure,function,and regulation of secretion[J].Physiological Reviews,2000,80(4):1523-1631.
[31]
ABDEL-NASER E M,HUSSIEN M F,EL-KHABAZ K A S.Some bacteriological studies on sub clinical mastitis in cattle and its relation to changes in the milk protein electrophoretic pattern[J].Assiut Veterinary Medical Journal,2010,56(127):58-74.
[32]
KESTER H J,SORTER D E,HOGAN J S.Activity and milk compositional changes following experimentally induced Streptococcus uberis bovine mastitis[J].Journal of Dairy Science,2015,98(2):999-1004.
[33]
MURPHY S C,CRANKER K,SENYK G F,et al.Influence of bovine mastitis on lipolysis and proteolysis in milk[J].Journal of Dairy Science,1989,72(3):620-626.
[35]
FARRELL H M,Jr.,MALIN E L,BROWN E M,et al.Review of the chemistry of αS2 -casein and the generation of a homologous molecular model to explain its properties[J].Journal of Dairy Science,2009,92(4):1338-1353.
[34]
SAEMAN A I,VERDI R J,GALTON D M,et al.Effect of mastitis on proteolytic activity in bovine milk[J].Journal of Dairy Science,1988,71(2):505-512.
[41]
REILING J H,SABATINI D M.Stress and mTORture signaling[J].Oncogene,2006,25(48):6373-6383.
[36]
SCHINDLER C,LEVY D E,DECKER T.JAK-STAT signaling:from interferons to cytokines[J].Journal of Biological Chemistry,2007,282(28):20059-20063.
[37]
COCOLAKIS E,DAI M O,DREVET L,et al.Smad signaling antagonizes STAT5-mediated gene transcription and mammary epithelial cell differentiation[J].Journal of Biological Chemistry,2008,283(3):1293-1307.
[38]
HARRIS J,STANFORD P M,SUTHERLAND K,et al.Socs2 and Elf5 mediate prolactin-induced mammary gland development[J].Molecular Endocrinology,2006,20(5):1177-1187.
[40]
ÅKERSTEDT M,WREDLE E,LAM V,et al.Protein degradation in bovine milk caused by Streptococcus agalactiae[J].Journal of Dairy Research,2012,79(3):297-303.
