金黄色葡萄球菌处理对奶牛乳腺上皮细胞外泌体表征的影响

doi:10.16431/j.cnki.1671-7236.2022.08.025

摘要/Abstract

摘要： 【目的】本研究旨在从金黄色葡萄球菌(Staphylococcus aureus,S.aureus,以下简称金葡菌)处理奶牛乳腺上皮细胞(bovine mammary epithelial cell,BMEC)上清中分离外泌体,探究金葡菌与BMEC数量比值(MOI)和处理后培养时间对外泌体总浓度的影响,并对金葡菌诱导BMEC释放的外泌体进行分离和鉴定。【方法】采用金葡菌临床分离菌株以MOI＝1和MOI＝10分别处理BMEC 3 h,对已处理的BMEC继续无外泌体培养9、12、24 h,同时设空白对照组,通过扫描电镜观察金葡菌处理对BMEC造成的超微结构损伤。采用超速离心法分离金葡菌诱导的BMEC上清液中的外泌体,分别用透射电镜、纳米颗粒追踪分析技术及Western blotting法对外泌体形态、颗粒大小和特异性标志蛋白进行分析鉴定。通过检测外泌体总蛋白浓度评估不同MOI和处理后培养时间对外泌体总量的影响,明确金葡菌诱导BMEC释放外泌体的最佳条件。【结果】通过扫描电镜观察发现,金葡菌处理BMEC后,细胞微绒毛脱落,细胞骨架破坏。透射电镜观察分离的外泌体为圆形的双层膜囊泡,直径在30~150 nm,形态均一。通过检测外泌体总蛋白浓度显示,在MOI＝10时,培养9、12和24 h金葡菌诱导BMEC释放外泌体蛋白浓度均高于MOI＝1,而培养12 h时释放外泌体蛋白浓度最高。对金葡菌诱导BMEC释放的外泌体进一步鉴定,其平均直径约为116 nm;Western blotting检测结果显示外泌体标记物CD9、CD81和TSG101表达阳性。【结论】本研究从形态学和分子生物学特征等方面证实获得的分离物为外泌体;金葡菌可以诱导BMEC释放外泌体,当MOI＝10时,金葡菌处理细胞3 h后继续无外泌体培养12 h,收获的细胞上清中外泌体蛋白含量最高。

关键词: 奶牛; 金黄色葡萄球菌; 乳腺上皮细胞; 外泌体

Abstract: 【Objective】 This study was aimed to isolate exosomes from bovine mammary epithelial cell (BMEC) infected with Staphylococcus aureus (S.aureus),and investigate the effects of MOI(S.aureus:BMEC) and time post infection on the total concentration of exosomes.Then,the exosomes induced by S.aureus were also isolated and identified.【Method】 BMEC were infected with two levels of S.aureus (MOI=1 and MOI=10) for 3 h,and further cultured for 9,12,and 24 h after the infection.While,a blank control group with no infection of S.aureus was also set up simultaneously.The damage of the ultrastructure of BMEC caused by S.aureus infection was observed by scanning electron microscope.The exosomes in the supernatant of S.aureus-infected BMEC were then isolated by ultracentrifugation.The morphology,particle size and marker proteins of the exosomes were analyzed by transmission electron microscopy,nanoparticle tracking analysis,and Western blotting,respectively.The total amount of exosomes at different MOI and post-infection time was evaluated by detecting the total protein concentration of exosomes.Consequently,the optimal conditions for S.aureus in inducing the release of exosomes from BMEC were determined.【Result】 Scanning electron microscope observation showed that S.aureus infection induced shedding of microvilli,and destruction of cytoskeleton of the BMEC.Exosomes were successfully isolated and purified from the supernatant of S.aureus-infected BMEC by ultracentrifugation.Transmission electron microscopy showed that the purified exosomes were spherical vesicles with a lipid bilayer membrane,and a diameter of 30-150 nm. The morphology of the exosomes in each group was uniform.The total protein concentration of exosomes induced by S.aureus.with MOI=10 was higher than that at MOI=1 at 9,12 and 24 h after the infection respectively, and it was the highest at 12 h after infection.Furthermore,nanoparticle tracking analysis detected the average diameter of exosomes induced by S.aureus was about 116 nm.Western blotting results showed that the exosome protein markers CD9,CD81,and TSG101 were positively expressed.【Conclusion】 The results indicated that the exosomes were successfully isolated in this study by inspecting their morphological and molecular biological characteristics.The releasing of the exosomes derived from BMEC could be induced by S.aureus.And the highest protein content in the exosomes derived from the BMEC was observed after treated by S.aureus for 3 h and cells cultured exosome-freely for 12 h with MOI=10.

Key words: dairy cows; Staphylococcus aureus; mammary epithelial cell; exosomes

中图分类号:

S858.23

蔡萌, 朱晓艳, 王梦玲, 刘子豪, 熊本海, 杨亮. 金黄色葡萄球菌处理对奶牛乳腺上皮细胞外泌体表征的影响[J]. 中国畜牧兽医, 2022, 49(8): 3091-3098.

CAI Meng, ZHU Xiaoyan, WANG Mengling, LIU Zihao, XIONG Benhai, YANG Liang. Effect of Staphylococcus aureus on Exosome Characterization of Mammary Epithelial Cells in Dairy Cows[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3091-3098.

参考文献

[1] HEIKKILA A M,LISKI E,PYORALA S,et al.Pathogen-specific production losses in bovine mastitis[J].Journal of Dairy Science,2018,101(10):9493-9504.
[2] ZHAO X,LACASSE P.Mammary tissue damage during bovine mastitis:Causes and control[J].Journal of Animal Science,2008,86(Suppl 13):57-65.
[3] 古丽努尔·哈森别克.奶牛乳房炎的危害、临床症状及防治措施[J].现代畜牧科技,2020,9:75-76. GULINUER H S B K.Hazards,clinical symptoms and prevention measures of dairy mastitis[J].Modern Animal Husbandry Technology,2020,9:75-76.(in Chinese)
[4] KRISHNAMOORTHY P,SURESH K P,JAYAMMA K S,et al.An understanding of the global status of major bacterial pathogens of milk concerning bovine mastitis:A systematic review and meta-analysis (scientometrics)[J].Pathogens,2021,10(5):545.
[5] AITKEN S L,CORL C M,SORDILLO L M.Immunopathology of mastitis:Insights into disease recognition and resolution[J].Journal of Mammary Gland Biology and Neoplasia,2011,16(4):291-304.
[6] MARJORIE B,JOHANN D,FRÉDÉRIC F,et al.Associations between properties linked with persistence in a collection of Staphylococcus aureus isolates from bovine mastitis[J].Veterinary Microbiology,2014,169(1-2):74-79.
[7] CONTRERAS G A,RODRÍGUEZ J M.Mastitis:Comparative etiology and epidemiology[J].Journal of Mammary Gland Biology and Neoplasia,2011,16(4):339-356.
[8] SINHA B,FRAUNHOLZ M.Staphylococcus aureus host cell invasion and post-invasion events[J].International Journal of Medical Microbiology,2010,300(2-3):170-175.
[9] CHRISTIAN G,WILLIAM L K.Staphylococcus aureus:New evidence for intracellular persistence[J].Trends in Microbiology,2008,17(2):59-65.
[10] HENG W,YUQI Z,QICHENG Z,et al.Staphylococcus aureus induces autophagy in bovine mammary epithelial cells and the formation of autophagosomes facilitates intracellular replication of Staph.aureus[J].Journal of Dairy Science,2019,102(9):8264-8272.
[11] RAGHU KALLURI V S L.The biology,function,and biomedical applications of exosomes[J].Science Letter,2020,6478(367):u6977.
[12] ZHANG X,XU D,SONG Y,et al.Research progress in the application of exosomes in immunotherapy[J].Frontiers in Immunology,2022,13:731516..
[13] KAKARLA R,HUR J,KIM Y J,et al.Apoptotic cell-derived exosomes:Messages from dying cells[J].Experimental and Molecular Medicine,2020,52(1):1-6.
[14] ZEMPLENI J,AGUILAR-LOZANO A,SADRI M,et al.Biological activities of extracellular vesicles and their cargos from bovine and human milk in humans and implications for infants[J].The Journal of Nutrition,2017,147(1):3-10.
[15] SUSUMU M,TATSURO H,ATARU K,et al.Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture[J].Journal of Animal Science and Biotechnology,2016,7(4):504-513.
[16] ZHANG M,MA Z,LI R,et al.Proteomic analysis reveals proteins and pathways associated with lactation in bovine mammary epithelial cell-derived exosomes[J].Journal of Proteome Research,2020,19(8):3211-3219.
[17] SOBUE A,ITO N,NAGAI T,et al.Astroglial major histocompatibility complex class Ⅰ following immune activation leads to behavioral and neuropathological changes[J].Glia,2018,66(5):1034-1052.
[18] MITCHELL M D,SCHOLZ-ROMERO K,REED S,et al.Plasma exosome profiles from dairy cows with divergent fertility phenotypes[J]. Journal of Dairy Science,2016,99(9):7590-7601.
[19] HIEMSTRA T F,CHARLES P D,GRACIA T,et al.Human urinary exosomes as innate immune effectors[J].Journal of the American Society of Nephrology,2014,25(9):2017-2027.
[20] MIYAKE H,LEE C,CHUSILP S,et al.Human breast milk exosomes attenuate intestinal damage[J].Pediatric Surgery International,2020,36(2):155-163.
[21] LARSSEN P,WIK L,CZARNEWSKI P,et al.Tracing cellular origin of human exosomes using multiplex proximity extension assays[J].Molecular and Cellular Proteomics,2017,16(3):502-511.
[22] MODEPALLI V,KUMAR A,HINDS L A,et al.Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)[J].BMC Genomics,2014,15:1012.
[23] PHAM C V,MIDGE S,BARUA H,et al.Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when'exosome-depleted serum'is utilised[J].Archives of Biochemistry and Biophysics,2021,708:108963.
[24] MITCHELL M D, SCHOLZ-ROMERO K,REED S,et al.Plasma exosome profiles from dairy cows with divergent fertility phenotypes[J]. National Agriculture Week,2016,9(99):7590-7601.
[25] SUN J,ASWATH K,SCHROEDER S G,et al.microRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection[J].BMC genomics,2015,16(1):806.
[26] CAI M,HE H,JIA X,et al.Genome-wide microRNA profiling of bovine milk-derived exosomes infected with Staphylococcus aureus[J].Cell Stress and Chaperones,2018,23(4):663-672.
[27] MA S,TONG C,IBEAGHA-AWEMU E M,et al.Identification and characterization of differentially expressed exosomal microRNAs in bovine milk infected with Staphylococcus aureus[J].BMC Genomics,2019,20(1):934.
[28] GENG N,WANG X,YU X,et al.Staphylococcus aureus avoids autophagy clearance of bovine mammary epithelial cells by impairing lysosomal function[J].Frontiers in Immunology,2020,11:746.
[29] STRICKLAND M,WATANABE S,BONN S M,et al.Tsg101/ESCRT-Ⅰ recruitment regulated by the dual binding modes of K63-linked diubiquitin[J].Structure,2022,30(2):289-299.
[30] CARRIERE J,BRETIN A,DARFEUILLE-MICHAUD A,et al.Exosomes released from cells infected with Crohn's disease-associated adherent-invasive Escherichia coli activate host innate immune responses and enhance bacterial intracellular replication[J].Inflammatory Bowel Diseases,2016,22(3):516-528.
[31] 赵文苹,王建发,范春玲.脂多糖刺激奶牛乳腺上皮细胞后外泌体的分离及鉴定[J].中国生物制品学杂志,2021,34(2):166-168. ZHAO W P,WANG J F,FAN C L.Isolation and identification of exosomes produced by stimulating mammary epithelial cells of dairy cows with lipopolysaccharide[J].Chinese Journal of Biologicals,2021,34(2):166-168.(in Chinese)
[32] 张天琦,许浩天,赵文苹,等.LPS处理奶牛乳腺上皮细胞后外泌体中差异miRNA分析[J].中国兽医学报,2021,41(12):2438-2444. ZHANG T Q,XU H T,ZHAO W P,et al.miRNA profiling of exosomes isolated from lipopolysaccharide-change dairy cow mammary epithelial cells[J].Chinese Journal of Veterinary Medicine,2021,41(12):2438-2444.(in Chinese)
[33] HUANG T,ZHOU C,CHE Y,et al.Exosomes derived from bovine mammary epithelial cells treated with transforming growth factor-beta1 inhibit the proliferation of bovine macrophages[J].Journal of Interferon and Cytokine Research,2019,39(12):752-759.
[34] BANNERMAN D D.Pathogen-dependent induction of cytokines and other soluble inflammatory mediators during intramammary infection of dairy cows[J].Journal of Animal Science.2009,87(13):10-25.
[35] GENINI S,BADAOUI B,SCLEP G,et al.Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources[J].BMC Genomics, 2011,12(1):225.
[36] RICH K A,BURKETT C,WEBSTER P.Cytoplasmic bacteria can be targets for autophagy[J].Cellular Microbiology,2003,5(7):455-468.
[37] LARABI A,DALMASSO G,DELMAS J,et al.Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn's disease-associated adherent-invasive E.coli[J].Gut Microbes,2020,11(6):1677-1694.
[38] KELLER M D,CHING K L,LIANG F X,et al.Decoy exosomes provide protection against bacterial toxins[J].Nature,2020,579(7798):260-264.