金黄色葡萄球菌PV-杀白细胞素的表达及其对奶牛乳腺上皮细胞的损伤作用

doi:10.11843/j.issn.0366-6964.2019.10.017

摘要/Abstract

摘要： 研究产PV-杀白细胞素（PVL）金黄色葡萄球菌ATCC49775、pvl缺陷株△pvl 49775和相应的回补株C-△pvl 49775以及体外重组的PVL（rPVL）对奶牛乳腺上皮细胞的损伤作用，为系统研究金黄色葡萄球菌感染奶牛乳腺上皮细胞的机制提供依据。采用原核表达的方法构建金黄色葡萄球菌LukS-PV和LukF-PV重组质粒并分别转化大肠杆菌BL21，诱导表达纯化后对其进行Western blot鉴定；用Live/Dead细胞荧光染色和Annexin V-FITC/PI双染法检测ATCC49775、△pvl 49775、C-△pvl 49775和rPVL对奶牛乳腺上皮细胞凋亡的影响。成功构建pET28a-LukS-PV、pET28a-LukF-PV重组表达载体，经诱导表达纯化后获得的LukS-PV浓度为0.6 mg·mL^-1，LukF-PV浓度为1.15 mg·mL^-1。rPVL主要诱导奶牛乳腺上皮细胞凋亡（P<0.01），金黄色葡萄球菌ATCC49775、△pvl 49775和C-△pvl 49775均能诱导奶牛乳腺上皮细胞明显凋亡和坏死，但pvl阳性菌株ATCC49775和C-△pvl 49775诱导的奶牛乳腺上皮细胞凋亡率和坏死率明显高于△pvl 49775（P<0.05）。结果表明原核表达的rPVL对奶牛乳腺上皮细胞表现出较强的毒性；pvl缺陷株△pvl 49775诱导奶牛乳腺上皮细胞凋亡和坏死的能力比pvl阳性菌株ATCC49775和C-△pvl 49775低，说明PVL在金黄色葡萄球菌感染奶牛乳腺上皮细胞过程中有重要作用。

Abstract: In order to provide a basis for systematically studying the mechanism of Staphylococcus aureus infection of bovine mammary epithelial cells, the damages of producing panton-valenine leukocidin (PVL) S. aureus ATCC49775, pvl-knockout mutant △pvl 49775, complemented mutant C-△pvl 49775 and recombinant PVL (rPVL) in vitro on bovine mammary epithelial cells were studied. Recombinant plasmids of S. aureus LukS-PV and LukF-PV were transformed into E. coli BL21, respectively. After expression and purification, they were identified by Western blot. The effects of ATCC49775, △pvl 49775, C-△pvl 49775 and rPVL on the apoptosis of bovine mammary epithelial cells were detected by Live/Dead cell fluorescence staining and Annexin V-FITC/PI double staining. The recombinant expression vectors pET28a-LukS-PV and pET28a-LukF-PV were successfully constructed. The concentration of LukS-PV obtained after induction and purification was 0.6 mg·mL^-1, and the concentration of LukF-PV was 1.15 mg·mL^-1. rPVL mainly induced apoptosis (P<0.01). Both S. aureus ATCC49775, △pvl 49775 and C-△pvl 49775 induced significant apoptosis and necrosis on bovine mammary epithelial cells, but ATCC49775 and C-△pvl 49775 induced worse (P<0.05). The results showed that rPVL of prokaryotic expression had a strong toxicity on bovine mammary epithelial cells. The pvl-knockout mutant △pvl 49775 induced the apoptosis and necrosis of the bovine mammary epithelial cells low than the pvl positive strain ATCC49775 and C-△pvl 49775. Our study indicates that the PVL plays an important role in S. aureus infected bovine mammary epithelial cells.

中图分类号:

S852.611

马维武, 周学章. 金黄色葡萄球菌PV-杀白细胞素的表达及其对奶牛乳腺上皮细胞的损伤作用[J]. 畜牧兽医学报, 2019, 50(10): 2105-2112.

MA Weiwu, ZHOU Xuezhang. Expression of Panton-Valenine Leukocidin from Staphylococcus aureus and Its Effects on Bovine Mammary Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2105-2112.

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参考文献 21

[1]	ZHANG X P, HU X M, RAO X C. Apoptosis induced by Staphylococcus aureus toxins[J]. Microbiol Res, 2017, 205:19-24.
[2]	VRIELING M, BOERHOUT E M, VAN WIGCHEREN G F, et al. LukMF' is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis[J]. Sci Rep, 2016, 6:37759.
[3]	FROMAGEAU A, CUNHA P, GILBERT F B, et al. Purified Staphylococcus aureus leukotoxin LukM/F'does not trigger inflammation in the bovine mammary gland[J]. Microb Pathog, 2011, 51(6):396-401.
[4]	王晓,俞英. 中国北方奶牛金葡菌乳房炎感染现状及耐药性和流行类型研究进展[J]. 畜牧兽医学报, 2015, 49(9):1477-1488.
	WANG X, YU Y. Infectious status of mastitis in dairy cattle induced by Staphylococcus aureus and its advances on epidemiological patterns and antimicrobial resistance in northern China[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 49(9):1477-1488. (in Chinese)
[5]	WANG W, LIN X H, JIANG T, et al. Prevalence and characterization of Staphylococcus aureus cultured from raw milk taken from dairy cows with mastitis in Beijing, China[J]. Front Microbiol, 2018, 9:1123.
[6]	崔新洁. 金黄色葡萄球菌诱导牛乳腺上皮细胞凋亡及其信号通路的研究[D]. 呼和浩特:内蒙古大学, 2013.
	CUI X J. The study on apoptosis and its signaling in bovine mammary epithelial cell induced by Staphylococcus aureus[D]. Hohhot:Inner Mongolia University, 2013. (in Chinese)
[7]	SEOL J W, KANG S J, PARK S Y. Silver ion treatment of primary cultured bovine mammary gland epithelial cell (BMEC) damage from Staphylococcus aureus-derived α-toxin[J]. Vet Res Commun, 2010, 34(1):33-42.
[8]	LIU Y X, CHEN W, ALI T, et al. Staphylococcal enterotoxin H induced apoptosis of bovine mammary epithelial cells in vitro[J]. Toxins (Basel), 2014, 6(12):3552-3567.
[9]	ALONZO Ⅲ F, TORRES V J. The bicomponent pore-forming leucocidins of Staphylococcus aureus[J]. Microbiol Mol Biol Rev, 2014, 78(2):199-230.
[10]	CHEN J, LUO Y P, ZHANG S, et al. Community-acquired necrotizing pneumonia caused by methicillin-resistant Staphylococcus aureus producing Panton-Valentine leukocidin in a Chinese teenager:case report and literature review[J]. Int J Infect Dis, 2014, 26:17-21.
[11]	HARCH S A J, MACMORRAN E, TONG S Y C, et al. High burden of complicated skin and soft tissue infections in the Indigenous population of Central Australia due to dominant Panton Valentine leucocidin clones ST93-MRSA and CC121-MSSA[J]. BMC Infect Dis, 2017, 17:405.
[12]	DUBOS M, BARRAUD O, FEDOU A L, et al. Prostatic abscesses and severe sepsis due to methicillin-susceptible Staphylococcus aureus producing Panton-Valentine leukocidin[J]. BMC Infect Dis, 2014, 14:466.
[13]	CHI C Y, LIN C C, LIAO I C, et al. Panton-Valentine leukocidin facilitates the escape of Staphylococcus aureus from human keratinocyte endosomes and induces apoptosis[J]. J Infect Dis, 2014, 209(2):224-235.
[14]	WOODIN A M. Purification of the two components of leucocidin from Staphylococcus aureus[J]. Biochem J, 1960, 75(1):158-165.
[15]	WOODIN A M. Fractionation of a leucocidin from Staphylococcus aureus[J]. Biochem J, 1959, 73(2):225-237.
[16]	常文娇, 马筱玲,沈继龙,等. Panton-Valentine杀白细胞素原核表达、纯化及生物学活性鉴定[J]. 安徽医科大学学报, 2011, 46(1):6-10.
	CHANG W J, MA X L, SHEN J L, et al. Prokaryotic expression and biological activity identification of the Panton-Valentine leukocidin of Staphylococcus aureus[J]. Acta Universitatis Medicinalis Anhui, 2011, 46(1):6-10. (in Chinese)
[17]	GENESTIER A L, MICHALLET M C, PRÉVOST G, et al. Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils[J]. J Clin Invest, 2005, 115(11):3117-3127.
[18]	蒋智鑫. 原代奶牛乳腺上皮细胞(pbMEC)的分离培养及亚硒酸钠对其炎性损伤的保护作用研究[D]. 雅安:四川农业大学, 2013.
	JIANG Z X. Isolation and culture of bovine mammary epithelial cells(pbMEC) and the protective effect of sodium selenite against inflammatory injury induced by LPS on the pbMEC[D]. Ya'an:Sichuan Agricultural University, 2013. (in Chinese)
[19]	LÖFFLER B, HUSSAIN M, GRUNDMEIER M, et al. Staphylococcus aureus Panton-Valentine leukocidin is a very potent cytotoxic factor for human neutrophils[J]. PLoS Pathog, 2010, 6(1):e1000715.
[20]	SPAAN A N, HENRY T, VAN ROOIJEN W J M, et al. The staphylococcal toxin Panton-Valentine leukocidin targets human C5a receptors[J]. Cell Host Microbe, 2013, 13(5):584-594.
[21]	TROMP A T, VAN GENT M, ABRIAL P, et al. Human CD45 is an F-component-specific receptor for the staphylococcal toxin Panton-Valentine leukocidin[J]. Nat Microbiol, 2018, 3(6):708-717.