抗菌肽Temprine-La（FS）抑制耐甲氧西林伪中间型葡萄球菌生物被膜形成的研究

doi:10.19640/j.cnki.jtau.2021.01.006

天津农学院学报 ›› 2021, Vol. 28 ›› Issue (1): 24-33.doi: 10.19640/j.cnki.jtau.2021.01.006

抗菌肽Temprine-La（FS）抑制耐甲氧西林伪中间型葡萄球菌生物被膜形成的研究

张欣, 潘晨浩, 赵瑞利^通信作者, 姜轩, 赵洋, 马吉飞, 于恩远, 李留安, 张振洲

天津农学院动物科学与动物医学学院,天津 300392

收稿日期:2020-06-29 出版日期:2021-03-31 发布日期:2021-04-02
通讯作者: 赵瑞利（1979—）,女,副教授,博士,研究方向:分子病原细菌学。E-mail:zhaoruili1109@126.com。
作者简介:张欣（1994—）,女,硕士在读,研究方向：分子病原细菌学。E-mail：1748850435@qq.com。
基金资助:
天津市应用基础与前沿技术研究计划一般项目（18JCYBJC30100,14JCYBJC30000）;天津市高校“中青年骨干创新人才”培养计划（无编号）;天津市“131”创新型人才培养计划（196500407A）;国家大学生创新创业项目（201910061015）

Inhibitory effect of antimicrobial peptide Temprine-La（FS）on biofilm formation of methicillin resistant Staphylococcus pseudintermedius

Zhang Xin, Pan Chenhao, Zhao Ruili^{Corresponding Author}, Jiang Xuan, Zhao Yang, Ma Jifei, Yu Enyuan, Li Liuan, Zhang Zhenzhou

College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China

Received:2020-06-29 Online:2021-03-31 Published:2021-04-02

摘要/Abstract

摘要： 为了探究抗菌肽Temprine-La（FS）对耐甲氧西林伪中间型葡萄球菌（MRSP）生物被膜形成的影响,本试验以Temprine-La为基序进行氨基酸改造合成新的抗菌肽（T-La（S）、T-La（FS）、RGD-T-La（S）、RGD-T-La（FS）以提高抗菌活性。通过微量稀释法测定多肽对MRSP生物被膜的最小抑菌浓度（MBIC）;结晶紫染色法（CV）检测多肽对MRSP生物被膜形成能力的影响;XTT法检测多肽对MRSP生物被膜代谢活性的影响、多肽对MRSP生物被膜胞外多糖含量的影响;扫描电镜（SEM）检测多肽对MRSP生物被膜形成的影响;荧光定量PCR法（q-PCR）分析多肽对MRSP生物被膜相关基因转录水平的影响。结果显示,T-La（S）、RGD-T-La（S）、T-La（FS）、RGD-T-La（FS）对MRSP的MBIC分别为：31.3、15.6、7.8、15.6 μg/mL;结晶紫染色法（CV）表明T-La（S）、RGD-T-La（S）、T-La（FS）、RGD-T-La（FS）对MRSP生物被膜形成有抑制作用;扫描电镜（SEM）结果显示多肽使MRSP生物被膜中细胞外基质大量减少,菌体裸露在物体表面;多肽可以显著降低MRSP生物被膜的代谢活性,抑制被膜菌的粘附作用;多肽能有效抑制MRSP生物被膜合成胞外多糖;荧光定量PCR结果表明,多肽作用后能够降低MRSP生物被膜相关基因的转录水平。因此,多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MRSP生物被膜的形成。

关键词: 抗菌肽Temprine-La（FS）, 耐甲氧西林伪中间型葡萄球菌（MRSP）, 生物被膜, 相关基因

Abstract: In order to investigate the effect of antimicrobial peptide Temprine-La（FS）on the biofilm formation of methicillin-resistant Staphylococcus pseudintermedius（MRSP）, we used Temprine-La as the motif to modify amino acids and synthesize new antimicrobial peptides（T-La（S）, T-La（FS）, RGD-T-La（S）, RGD-T-La（FS）to improve the antibacterial activity. The minimal inhibitory concentration（MBIC）of peptides on MRSP biofilm was determined by microdilution method; The ability of peptides to form MRSP biofilm was detected by crystal violet staining（CV）; The metabolic activity of MRSP biofilm was detected by XTT method; The content of extracellular polysaccharide in MRSP biofilm was detected by polypeptides; the effect of polypeptides on the formation of MRSP biofilm was detected by scanning electron microscopy（SEM）; Q-PCR was used to analyze the effect of polypeptides on the transcription level of MRSP biofilm related genes. The results showed that the MBIC of T-La（S）, RGD-T-La（S）, T-La（FS）and RGD-T-La（FS）to MRSP were 31.3, 15.6, 7.8 and 15.6 μg/mL, respectively; Crystal violet staining（CV）showed that T-La（S）, RGD-T-La（S）, T-La（FS）and RGD-T-La（FS）inhibited the formation of MRSP biofilm; The results of scanning electron microscopy（SEM）showed that polypeptides significantly reduced the extracellular matrix of MRSP biofilm and exposed the bacteria on the surface of the object; Polypeptides could significantly reduce the metabolic activity of MRSP biofilm and inhibit the adhesion of the biofilm; Polypeptides could effectively inhibit the synthesis of extracellular polysaccharides from MRSP biofilm; The results of fluorescent quantitative PCR showed that polypeptides could reduce the transformation of genes related to MRSP biofilm Recording level. Therefore, polypeptides mainly inhibit the formation of MRSP biofilm by affecting the transcription level of biofilm related genes and blocking the synthesis and secretion of extracellular polysaccharides.

Key words: antimicrobial peptide Temprine-La（FS）, methicillin resistant Staphylococcus pseudintermedius （MRSP）, biofilm, related genes

中图分类号:

S816.7

张欣, 潘晨浩, 赵瑞利, 姜轩, 赵洋, 马吉飞, 于恩远, 李留安, 张振洲. 抗菌肽Temprine-La（FS）抑制耐甲氧西林伪中间型葡萄球菌生物被膜形成的研究[J]. 天津农学院学报, 2021, 28(1): 24-33.

Zhang Xin, Pan Chenhao, Zhao Ruili, Jiang Xuan, Zhao Yang, Ma Jifei, Yu Enyuan, Li Liuan, Zhang Zhenzhou. Inhibitory effect of antimicrobial peptide Temprine-La（FS）on biofilm formation of methicillin resistant Staphylococcus pseudintermedius[J]. Journal of Tianjin Agricultural University, 2021, 28(1): 24-33.

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http://xuebao.tjau.edu.cn/CN/Y2021/V28/I1/24

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抗菌肽Temprine-La（FS）抑制耐甲氧西林伪中间型葡萄球菌生物被膜形成的研究

Inhibitory effect of antimicrobial peptide Temprine-La（FS）on biofilm formation of methicillin resistant Staphylococcus pseudintermedius

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