抗菌肽Temprine-La(FS)抑制2型猪链球菌生物被膜形成的研究

doi:10.16431/j.cnki.1671-7236.2021.01.036

摘要/Abstract

摘要： 试验旨在研究抗菌肽Temprine-La（S）（T-La（S））、Temprine-La（FS）（T-La（FS））、RGD-T-La（S）和RGD-T-La（FS）对2型猪链球菌（SS2）生物被膜形成的抑制作用。通过结晶紫染色法（CV）检测SS2生物被膜形成能力；微量稀释法测定抗菌肽对SS2生物被膜的最小生物被膜抑菌浓度（MBIC）和最小生物被膜杀菌浓度（MBEC）；结晶紫染色法和扫描电镜（SEM）检测抗菌肽对SS2生物被膜形成的影响；XTT法检测抗菌肽对SS2生物被膜代谢活性的影响；苯酚硫酸法检测抗菌肽对SS2生物被膜胞外多糖含量的影响；建立猪链球菌-斑马鱼感染模型，HE染色法观察T-La（FS）对斑马鱼脑组织病理变化的影响；实时荧光定量PCR法分析抗菌肽对SS2生物被膜相关基因及对斑马鱼炎性细胞因子基因转录水平的影响。结果显示，SS2具有良好的生物被膜形成能力；T-La（S）、RGD-T-La（S）、T-La（FS）和RGD-T-La（FS）对SS2生物被膜的MBIC分别为31.3、15.6、7.8和15.6 μg/mL；MBEC分别为62.6、31.2、15.6和31.2 μg/mL；结晶紫染色结果表明，抗菌肽对SS2生物被膜的形成有抑制作用；扫描电镜结果显示，抗菌肽使SS2生物被膜中的细菌数量和生物被膜的形态发生明显变化，细胞外基质大量减少；XTT法结果显示，抗菌肽可显著降低SS2生物被膜的代谢活性；苯酚硫酸法结果显示，抗菌肽能有效抑制SS2生物被膜合成胞外多糖；实时荧光定量PCR结果表明，抗菌肽作用后降低了SS2生物被膜基因的转录水平；T-La（FS）作用后TLR2、MyD88及促炎性细胞因子基因的转录水平显著或极显著降低（P<0.05；P<0.01），抗炎性细胞因子基因的转录水平显著或极显著升高（P<0.05；P<0.01）。抗菌肽主要通过影响生物被膜相关基因转录水平和阻断胞外多糖的合成与分泌来抑制SS2生物被膜的形成，其中T-La（FS）可能通过抑制TLR2信号通路中TLR2和MyD88分子表达，抑制炎性细胞因子的释放，减轻脑膜炎的炎性反应。

关键词: 抗菌肽Temprine-La(FS); 2型猪链球菌; 生物被膜; 斑马鱼; TLR2; MyD88

Abstract: The aim of this study was to investigate the inhibitory effects of antimicrobial peptides Temprine-La(S)(T-La(S)),Temprine-La(FS) (T-La(FS)),RGD-T-La(S) and RGD-T-La(FS) on biofilm formation of Streptococcus suis type 2 (SS2).The biofilm formation ability of SS2 was detected by crystal violet staining (CV).The MBIC and MBEC of antimicrobial peptides against SS2 biofilm were determined by microdilution method.Crystal violet staining and scanning electron microscopy (SEM) were used to detect the effect of antimicrobial peptides on biofilm formation of SS2.XTT method was used to detect the effect of antimicrobial peptides on the biofilm metabolic activity of SS2.Phenol sulfuric acid method was used to detect the effect of antimicrobial peptides on the extracellular polysaccharide content of SS2 biofilm.HE staining was used to observe the effect of T-La(FS) on the pathological changes of zebrafish brain tissue.The effects of antimicrobial peptides on the biofilm related genes of SS2 and the transcription levels of inflammatory cytokines genes in zebrafish were analyzed by Real-time PCR.The results showed that SS2 had good biofilm formation ability.The MBIC of T-La(S),RGD-T-La(S),T-La(FS) and RGD-T-La(FS) on the biofilm of SS2 were 31.3,15.6,7.8 and 15.6 μg/mL,respectively.MBEC were 62.6,31.2,15.6 and 31.2 μg/mL,respectively.The results of crystal violet staining showed that the antibacterial peptides had inhibitory effect on the biofilm formation of SS2.The results of SEM showed that the number of bacteria and the morphology of biofilm of SS2 were significantly changed by antimicrobial peptides,and the extracellular matrix was greatly reduced.The results of XTT showed that antimicrobial peptides could significantly reduce the metabolic activity of the biofilm of SS2.The results of phenol sulfuric acid method showed that antimicrobial peptides could effectively inhibit the synthesis of extracellular polysaccharide from the biofilm of SS2.The results of Real-time PCR showed that the antibacterial peptide reduced the transcription level of biofilm gene of SS2.After treatment with T-La(FS),the transcription levels of TLR2,MyD88 and pro-inflammatory cytokine genes were significantly or extremely significantly decreased (P<0.05;P<0.01),while the transcription levels of anti-inflammatory cytokine genes were significantly or extremely significantly increased (P<0.05;P<0.01).Antimicrobial peptides inhibited the biofilm formation of SS2 mainly by affecting the transcription level of biofilm related genes and blocking the synthesis and secretion of extracellular polysaccharides.T-La (FS) might inhibit the release of inflammatory cytokines and alleviate the inflammatory response of meningitis by inhibiting the expression of TLR2 and MyD88 molecules in TLR2 signaling pathway.

Key words: antimicrobial peptide Temprine-La(FS); Streptococcus suis type 2; biofilm; zebrafish; TLR2; MyD88

中图分类号:

S816.7

张欣, 潘晨浩, 赵瑞利, 姜轩, 金天明, 赵洋, 马吉飞, 于恩远, 李留安, 张振洲, 李桂霞, 李瑞忠. 抗菌肽Temprine-La(FS)抑制2型猪链球菌生物被膜形成的研究[J]. 中国畜牧兽医, 2021, 48(1): 324-337.

ZHANG Xin, PAN Chenhao, ZHAO Ruili, JIANG Xuan, JIN Tianming, ZHAO Yang, MA Jifei, YU Enyuan, LI Liuan, ZHANG Zhenzhou, LI Guixia, LI Ruizhong. Inhibitory Effect of Antimicrobial Peptide Temprine-La(FS) on Biofilm Formation of Streptococcus suis Type 2[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 324-337.

参考文献

[1] 刘琪,王娟,周月如,等.广东地区健康猪群和发病猪群猪链球菌流行病学调查分析[J].中国畜牧兽医,2017,44(6):1825-1831. LIU Q,WANG J,ZHOU Y R,et al.The Streptococcus suis epidemiological analysis of healthy and infected swine in Guangdong province[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1825-1831.(in Chinese)
[2] ZHANG Y Y,DING D D,LIU M L,et al.Effect of the glycosyltransferases on the capsular polysa-ccharide synthesis of Streptococcus suis serotype 2[J].Microbiological Research,2016,185:45-54.
[3] 尤理想,赵青,周敏,等.细菌生物膜检测方法改进与应用[J].实验技术与管理,2015,32(3):72-76. YU L X,ZHAO Q,ZHOU M,et al.Improved method of bacterial biofilm detection and its application[J].Experimental Technology and Management,2015,32(3):72-76.(in Chinese)
[4] CASTELO-BRANCO D S C M,RIELLO G B,VASCONCELOS D C,et al.Farnesol increases the susceptibility of Burkholderia pseudomallei biofilm to antimicrobials used to treat melioidosis[J].Journal of Applied Microbiology,2016,120(3):600-606.
[5] 王燕,陈立志,刘晓颖,等.以斑马鱼为模式动物的病原微生物感染模型的研究进展[J].吉林农业大学学报,2010,32(S1):49-52. WANG Y,CHEN L Z,LIU X Y,et al.Progress on infectious models of pathogenic microorganism using Danio rerio as a model animal[J].Journal of Jilin Agricultural University,2010,32(S1):49-52.(in Chinese)
[6] LI S,HAO L,BAO W,et al.A novel short anionic antibacterial peptide isolated from the skin of Xenopus laevis with broad antibacterial activity and inhibitory activity against breast cancer cell[J].Archives of Microbiology,2016,198(5):473-482.
[7] 张福梅,李明生.抗菌肽在免疫防御和抗肿瘤中的作用[J].国外医药抗生素分册,2017,38(6):284-289. ZHANG F M,LI M S.The role of antimicrobial peptides in immune defense and anti-tumor[J].World Notes on Antibiotics,2017,38(6):284-289.(in Chinese)
[8] 回丽媛,李军,朱明星,等.抗菌肽17BIPHE2对金黄色葡萄球菌生物被膜的抑制作用[J].微生物学通报,2017,44(4):872-880. HUI L Y,LI J,ZHU M X,et al.Inhibition of Staphylococcus aureus biofilm by antimicrobial peptide 17BIPHE2[J].Microbiology China,2017,44(4):872-880.(in Chinese)
[9] 刘梦月,刘珊珊,付超,等.新牛蛙抗肿瘤肽RGD-T-La(FS)嵌合体的设计及其抗肿瘤作用[J].中国兽医学报,2018,38(1):39-50. LIU M Y,LIU S S,FU C,et al.Design and antitumor activity of novel antitumor peptide RGD-T-La (FS) chimera from Catesbeiana[J].Chinese Journal of Veterinary Science,2018,38(1):39-50.(in Chinese)
[10] 于文会,许晶,魏庆微,等.蒲公英水提物对猪链球菌生物被膜体外干预作用[J].畜牧兽医学报,2015,46(10):1875-1881. YU W H,XU J,WEI Q W,et al.Dandelion aqueous extract affects biofilm formation by S.suis[J].Chinese Journal of Animal and Veterinary Sciences,2015,46(10):1875-1881.(in Chinese)
[11] 吴静,陈瑛,贾桂珍.中草药对猪链球菌生物被膜形成的影响[J].畜牧与兽医,2015,47(1):106-108. WU J,CHEN Y,JIA G Z.Effect of Chinese herbal medicine on biofilm formation of Streptococcus suis[J].Animal Husbandry & Veterinary Medicine,2015,47(1):106-108.(in Chinese)
[12] 姚孟霞,张菁芸,陈锦萍,等.番木瓜籽挥发油对变异链球菌的抑制作用研究[J].食品研究与开发,2019,40(7):64-68. YAO M X,ZHANG J Y,CHEN J P,et al.The antibacteria effect of Carica papaya Linn.seed essential oil on Streptococcus mutans[J].Food Research and Development,2019,40(7):64-68.(in Chinese)
[13] 刘冰,韩强,盛尊来,等.黄连水提物盐酸小檗碱及黄连碱对猪链球菌生物被膜的体外干预作用[J].中国兽医杂志,2015,51(4):16-19. LIU B,HAN Q,SHENG Z L,et al.Intervention effects of water extract,berberine hydrochloride and coptisine from Rhizoma Coptis against Streptococcus suis biofilm in vitro[J].Chinese Journal of Veterinary Medicine,2015,51(4):16-19.(in Chinese)
[14] DA SILVA W J,SENEVIRATEN J,PARAHITIYAWA N,et al.Improvement of XTT assay performance for studies involving Candida albicans biofilms[J].Brazilian Dental Journal,2008,19(4):364-369.
[15] DI D E G,CAVALLO I,GUEMBE M,et al.The clinical biofilm ring test:A promising tool for the clinical assessment of biofilm-producing Candida species[J].FEMS Yeast Research,2018,18(3):foy025.
[16] 刘玉红.磺化壳聚糖对细菌及其生物被膜抑制作用的研究[D].杭州:浙江工商大学,2019. LIU Y H.The inhibiting effects of sulfonated chitosan on bacteria and its biofilm[D].Hangzhou:Zhejiang Gongshang University,2019.(in Chinese)
[17] 靳胜男,舒慧萍,张冬星,等.被膜态与浮游态猪源乳杆菌上清液抑菌活性比较分析[J].中国畜牧兽医,2019,46(11):3358-3369. JIN S N,SHU H P,ZHANG D X,et al.Comparative analysis of the antimicrobial activity of Lactobacillus supernatants from swine in biofilm and plankton[J].China Animal Husbandry & Veterinary Medicine,2019,46(11):3358-3369.(in Chinese)
[18] 刘冰.黄连及其单体对S.suis生物被膜luxS基因及毒力因子的影响[D].哈尔滨:东北农业大学,2014. LIU B.Effect of Rhizoma coptidis and its monomers on the luxS gene and virulence genes against Streptococcus suis biofilm[D].Harbin:Northeast Agricultural University,2014.(in Chinese)
[19] WANG Y,WANG Y X,LIU B B,et al.pdh modulate virulence through reducing stress tolerance and biofilm formation of Streptococcus suis serotype 2[J].Virulence,2019,10(1):588-599.
[20] YANG Y B,WANG S,WANG C,et al.Emodin affects biofilm formation and expression of virulence factors in Streptococcus suis ATCC700794[J].Archives of Microbiology,2015,197(10):1173-1180.
[21] 熊关庆,冯杨,杨玉涔,等.厚朴酚对斑马鱼(Danio rerio)的急性毒性病理损伤评估[J].四川农业大学学报,2020,38(1):105-112. XIONG G Q,FENG Y,YANG Y C,et al.Evaluation of acute toxicologic pathology following immersion of honokiol in zebrafish (Danio rerio)[J].Journal of Sichuan Agricultural University,2020,38(1):105-112.(in Chinese)
[22] 郑鑫.斑马鱼非经典炎症小体调控抗感染和内毒素休克效应[D].上海:华东理工大学,2018. ZHENG X.Role of zebrafish caspy2-mediated non-canonical inflammsome activation in anti-infection and endotoxic shock[D].Shanghai:East China University of Science and Technology,2018.(in Chinese)
[23] 阳大海.胞内细菌性感染激活模式识别受体信号介导的宿主免疫应答机制[D].上海:华东理工大学,2015. YANG D H.Host immune responses to intracellular bacterial infection mediated by activation pattern recognition receptor signaling pathway[D].Shanghai:East China University of Science and Technology,2015.(in Chinese)
[24] 刘晓红.两种重要鱼类致病菌感染及减毒活疫苗接种途径的研究[D].上海:华东理工大学,2015. LIU X H.Portal of entry for two pathogens and a live attenuated vaccine into fish[D].Shanghai:East China University of Science and Technology,2015.(in Chinese)
[25] 丁云超.鱼类卵黄高磷蛋白衍生抗菌肽的免疫调节功能的研究[D].青岛:中国海洋大学,2012. DING Y C.Study on the immunomodulatory activity of phosvitin-derived antibacterial peptide in zebrafish[D].Qingdao:Ocean University of China,2012.(in Chinese)
[26] SÁNCHEZ-GÓMEZ S,FERRER-ESPADA R,STEWART P S,et al.Antimicrobial activity of synthetic cationic peptides and lipopeptides derived from human lactoferricin against Pseudomonas aeruginosa planktonic cultures and biofilms[J].BMC Microbiology,2015,15(1):137.
[27] XIE T,LIAO Z,LEI H,et al.Antibacterial activity of food-grade chitosan against Vibrio parahaemolyticus biofilms[J].Microbial Pathogenesis,2017,110:291-297.
[28] 汪洋.猪链球菌生物被膜形成及致病机理研究[D].南京:南京农业大学,2011. WANG Y.Study on the mechanism of biofilm formation and molecular pathogenesis of Streptococcus suis[D].Nanjing:Nanjing Agricultural University,2011.(in Chinese)
[29] NEELY M N,PFEIFER J D,CAPARON M.Streptococcus-zebrafish model of bacterial patho-genesis[J].Infection and Immunity,2002,70(7):3904-3914.
[30] 周谦,覃美娜,陈硕琳,等.变形链球菌提取物对人牙髓细胞TOLL样受体2表达的影响[J].广西医科大学学报,2018,35(6):770-773. ZHOU Q,QIN M N,CHEN S L,et al.Effect of extracts from Streptococcus mutans on the expression of Toll-like receptor 2 in human dental pulp cells[J].Journal of Guangxi Medical University,2018,35(6):770-773.(in Chinese)
[31] KUO A H,SCHEEREN F A.Cell-intrinsic TLR2/Myd88 pathway in breast and colon cance[J].Cell Cycle,2014,13(24):3785-3786.