鸢尾黄素作为MgrA抑制剂对小鼠金黄色葡萄球菌肺炎的治疗作用

doi:10.16431/j.cnki.1671-7236.2021.07.038

摘要/Abstract

摘要： 本研究旨在从中药小分子库中筛选出金黄色葡萄球菌（Staphylococcus aureus）转录调控因子MgrA的单体抑制剂，并探讨其对金黄色葡萄球菌主要毒力因子的影响及对小鼠金黄色葡萄球菌肺炎的治疗作用。使用荧光各向异性分析法进行抑制剂的筛选，通过实时荧光定量PCR、溶血试验及纤维蛋白原黏附试验考察药物对MrgA调控的相关毒力基因转录表达及对溶血和黏附的抑制作用，采用热稳定迁移试验对其抑制机制进行初步探讨，最后通过建立小鼠肺炎模型评估鸢尾黄素对金黄色葡萄球菌诱导的小鼠肺炎的治疗作用。结果显示，鸢尾黄素作为MgrA的抑制剂，在不影响细菌生长的低浓度下（IC₅₀=20.35 μg/mL）就能显著抑制MgrA的活性（P<0.05）；实时荧光定量PCR结果显示，鸢尾黄素可显著降低fnba基因的转录水平（P<0.05），极显著降低hla和RNAⅢ基因的转录水平（P<0.01），同时极显著上调ebh、srap和spa基因的转录水平（P<0.01）；溶血试验显示，8 μg/mL鸢尾黄素可极显著抑制USA300菌株的溶血作用（P<0.01）；纤维蛋白原黏附试验证实32 μg/mL鸢尾黄素可极显著抑制USA300菌株的黏附活性（P<0.01）；热稳定迁移试验揭示了鸢尾黄素是通过与MgrA结合从而抑制其活性。小鼠肺炎试验结果显示，鸢尾黄素能极显著提高金黄色葡萄球菌感染小鼠的存活率（P<0.01），极显著减少其肺脏载菌量（P<0.01），并减轻小鼠肺脏组织的病理损伤和炎症反应。以上结果表明，鸢尾黄素能通过抑制转录调控因子MgrA的活性对金黄色葡萄球菌感染引起的小鼠肺炎起到治疗作用，可作为开发治疗金黄色葡萄球菌感染的先导化合物，并为以毒力因子MgrA为靶标的药物研发提供了理论依据。

关键词: 鸢尾黄素; 金黄色葡萄球菌; MgrA; 抑制剂; 小鼠肺炎

Abstract: The purpose of this study was to screen out the monomer inhibitors of Staphylococcus aureus transcriptional regulator MgrA from the small molecule library of traditional Chinese medicine,and explore its effect on the main virulence factors of Staphylococcus aureus and its therapeutic effect on Staphylococcus aureus-induced pneumonia in mice.Fluorescence anisotropy analysis method was used to screen inhibitors,Real-time PCR,hemolysis test and fibrinogen adhesion test were used to investigate the transcriptional expression of related virulence genes regulated by MrgA and the inhibitory effect on hemolysis and adhesion.The thermal stable migration experiment was used to preliminarily explore its inhibitory mechanism.Finally,a mouse pneumonia model was established to evaluate the therapeutic effect of Tectorigenin on Staphylococcus aureus-induced pneumonia in mice.The results showed that Tectorigenin,as an inhibitor of MgrA,could significantly inhibit the activity of MgrA at a low concentration (IC₅₀=20.35 μg/mL) that did not affect bacterial growth (P<0.05).Real-time PCR results proved that Tectorigenin significantly reduced the transcription levels of fnba gene (P<0.05), extremely significantly reduced the transcription levels of hla and RNAⅢ genes (P<0.01),and at the same time extremely significantly increased the transcription levels of ebh, srap and spa genes (P<0.01).Hemolysis experiments showed that Tectorigenin could extremely significantly inhibit the hemolysis of USA300 strain at a concentration of 8 μg/mL (P<0.01).Fibrinogen adhesion experiments confirmed that Tectorigenin extremely significantly inhibited the adhesion activity of USA300 strain at 32 μg/mL (P<0.01).Thermal shift assay revealed that Tectorigenin inhibited its activity by binding to MgrA.The results of mouse pneumonia experiments showed that Tectorigenin could extremely significantly improve the survival rate of mice infected with Staphylococcus aureus (P<0.01),reduce the amount of bacteria in their lungs (P<0.01), and reduce the pathological damage and inflammation of lung tissue in mouse.The above results indicated that Tectorigenin could treat mouse pneumonia caused by Staphylococcus aureus by inhibiting the activity of the transcriptional regulator MgrA.Tectorigenin could be used as a lead compound for the development of treatment of Staphylococcus aureus infection,and provided a theoretical basis for the development of drugs targeting MgrA.

Key words: Tectorigenin; Staphylococcus aureus; MgrA; inhibitor; mice pneumonia

中图分类号:

S853.7

苏立燕, 王莉, 吴佳岂, 关舒函, 王大成, 王琳. 鸢尾黄素作为MgrA抑制剂对小鼠金黄色葡萄球菌肺炎的治疗作用[J]. 中国畜牧兽医, 2021, 48(7): 2617-2626.

SU Liyan, WANG Li, WU Jiaqi, GUAN Shuhan, WANG Dacheng, WANG Lin. Therapeutic Effect of Tectorigenin as MgrA Inhibitor on Staphylococcus aureus-induced Pneumonia in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2617-2626.

参考文献

[1] 郭英,滕达,王秀敏,等.抗菌肽NZ2114对奶牛乳房炎源金黄色葡萄球菌细胞膜、基因组和耐药性的影响[J].中国畜牧兽医,2019,46(4):1181-1190. GUO Y,TENG D,WANG X M,et al.Effects of antimicrobial peptide NZ2114 on cell membrane,genome and drug resistance of Staphylococcus aureus from dairy cow mastitis[J].China Animal Husbandry & Veterinary Medicine,2019,46(4):1181-1190.(in Chinese)
[2] 霍明飞,王鑫,戴小寒,等.亚抑菌浓度穿心莲提取物对减缓金黄色葡萄球菌α-溶血素介导的A549细胞损伤作用的影响[J].中国畜牧兽医,2013,40(5):110-114. HUO M F,WANG X,DAI X H,et al.Effects of Andrographis paniculata extract with sub-inhibitory concentration on the reduction of Staphylococcus aureus α-hemolysin-mediated A549 cell damage[J].China Animal Husbandry & Veterinary Medicine,2013,40(5):110-114.(in Chinese)
[3] ABDO M S A,KAI X,ZHONGGAI C,et al.Tectorigenin alleviates inflammation,apoptosis,and ossification in rat tendon-derived stem cells via modulating NF-kappa B and MAPK pathways[J].Frontiers in Cell and Developmental Biology,2020,8:568894.
[4] JENUL C,HORSWILL A R.Regulation of Staphylo-coccus aureus virulence[J].Microbiology Spectrum,2019,7(2):GPP3-0031-2018.
[5] NOVICK R P.Autoinduction and signal transduction in the regulation of staphylococcal virulence[J].Molecular Microbiology,2003,48(6):1429-1449.
[6] 廖明喻,逄龙,杨娅丽,等.金黄色葡萄球菌主要毒力因子对宿主固有免疫系统的影响[J].微生物学杂志,2015,35(5):102-107. LIAO M Y,PANG L,YANG Y L,et al.Effects of the main virulence factors of Staphylococcus aureus on the host innate immune system[J].Chinese Journal of Microbiology,2015,35(5):102-107.(in Chinese)
[7] INGAVALE S,VAN WAMEL W,LUONG T T,et al.Rat/MgrA,a regulator of autolysis,is a regulator of virulence genes in Staphylococcus aureus[J].Infection and Immunity,2005,73(3):1423-1431.
[8] CHEN P R,BAE T,WILLIAMS W A,et al.An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus[J].Nature Chemical Biology,2006,2(11):591-595.
[9] 刘彩林,明亮.异质性万古霉素中介金黄色葡萄球菌的流行性及mgrA基因对万古霉素耐药性影响的研究[J].中国抗生素杂志,2020,45(2):175-180. LIU C L,MING L.Study on the prevalence of heterogeneous vancomycin-mediated Staphylococcus aureus and the effect of mgrA gene on vancomycin resistance[J]. Chinese Journal of Antibiotics,2020,45(2):175-180.(in Chinese)
[10] ZHANG H,JIANG J M,HAN L,et al.Uncariitannin,a polyphenolic polymer from Uncaria gambier,attenuates Staphylococcus aureus virulence through an MgrA-mediated regulation of alpha-hemolysin[J].Pharmacological Research,2019,147:104328.
[11] SUN F,ZHOU L,ZHAO B C,et al.Targeting MgrA-mediated virulence regulation in Staphylococcus aureus[J].Cell Chemical Biology,2011,18(8):1032-1041.
[12] YAO X,LI K,LIANG C,et al.Tectorigenin enhances PDX1 expression and protects pancreatic β-cells by activating ERK and reducing ER stress[J].The Journal of Biological Chemistry,2020,295(37):12975-12992.
[13] RIORDAN J T,MUTHAIYAN A,VAN VOORHIES W,et al.Response of Staphylococcus aureus to salicylate challenge[J].Journal of Bacteriology,2007,189(1):220-227.
[14] CROSBY H A,SCHLIEVERT P M,MERRIMAN J A,et al.The Staphylococcus aureus global regulator MgrA modulates clumping and virulence by controlling surface protein expression[J].PLoS Pathogens,2016,12(5):e1005604.
[15] KWIECINSKI J M,CROSBY H A,VALOTTEAU C,et al.Staphylococcus aureus adhesion in endovascular infections is controlled by the ArlRS-MgrA signaling cascade[J].PLoS Pathogens,2019,15(5):e1007800.
[16] ZHANG H,LUAN Y,JING S,et al.Baicalein mediates protection against Staphylococcus aureus-induced pneumonia by inhibiting the coagulase activity of vWbp[J].Biochemical Pharmacology,2020,178:114024.
[17] MU D,LUAN Y,WANG L,et al.The combination of salvianolic acid A with latamoxef completely protects mice against lethal pneumonia caused by methicillin-resistant Staphylococcus aureus[J].Emerging Microbes Infections,2020,9(1):169-179.
[18] JIN Z,JIANG Q,FANG B,et al.The ArlR-MgrA regulatory cascade regulates PIA-dependent and protein-mediated biofilm formation in Rbf-dependent and Rbf-independent pathways[J].International Journal of Medical Microbiology,2019,309(2):85-96.
[19] LEI M G,LEE C Y.MgrA activates staphylococcal capsule via sIgA-dependent promoter[J].Journal of Bacteriology,2020,203(2):e00495-20.
[20] BO G,YU M,LITAO Z,et al.Identification and characterization of the chemical components of Iris tectorum Maxim.and evaluation of their nitric oxide inhibitory activity[J].Rapid Communications in Mass Spectrometry,2021,15;35(1):e8959.
[21] GAO Z,LUAN Y,YANG P,et al.Targeting staphylocoagulase with isoquercitrin protects mice from Staphylococcus aureus-induced pneumonia[J].Applied Microbiology and Biotechnology,2020,104(9):3909-3919.
[22] JIANG Q,JIN Z,SUN B.MgrA negatively regulates biofilm formation and detachment by repressing the expression of psm operons in Staphylococcus aureus[J].Applied and Environmental Microbiology,2018,84(16):e01008-18.
[23] MCADOW M,MISSIAKAS D M,SCHNEEWIND O.Staphylococcus aureus secretes coagulase and von Willebrand factor binding protein to modify the coagulation cascade and establish host infections[J].Journal of Innate Immunity,2012,4(2):141-148.
[24] KR R,GUPTA,THANH T,et al.RNAⅢ of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(45):14036-14041.
[25] LEI M G,GUDETA D D,LUONG T T,et al.MgrA negatively impacts Staphylococcus aureus invasion by regulating capsule and FnbA[J].Infection and Immunity,2019,87(12):e00590-19.
[26] LI L,WANG G,CHEUNG A,et al.MgrA governs adherence,host cellinteraction,and virulence in a murine model of bacteremia due to Staphylococcus aureus[J].Journal of Infectious Diseases,2019,220(6):1019-1028.