毛蕊花糖苷抑制2型猪链球菌的溶血素蛋白活性而降低其小鼠致病性

doi:10.3969/j.issn.1004-1524.2022.08.05

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (8): 1609-1616.DOI: 10.3969/j.issn.1004-1524.2022.08.05

毛蕊花糖苷抑制2型猪链球菌的溶血素蛋白活性而降低其小鼠致病性

詹佳飞¹(), 徐魁², 张磊¹, 夏介英¹, 洪杨¹, 董涵¹, 刘洋露¹, 周静¹, 袁明铭¹, 王永金¹, 鄢良春¹^,^*()

1.四川省中医药科学院实验动物中心,四川成都 610041
2.四川农业大学动物医学院,四川成都 611130

收稿日期:2021-12-22 出版日期:2022-08-25 发布日期:2022-08-26
通讯作者: 鄢良春
作者简介:*鄢良春,E-mail: 419746244@qq.com
詹佳飞(1992—),女,山西太原人,硕士研究生,主要从事动物传染病学研究。E-mail: zhanjiafei@hotmail.com
基金资助:
四川省省级科研院所基本科研业务费(A-2021N-14);四川省中医药管理局科学技术研究专项(A-2021-29)

Verbascoside lowers Streptococcus suis serotype 2 pathogenicity in mice by inhibiting hemolytic activity of suilysin

ZHAN Jiafei¹(), XU Kui², ZHANG Lei¹, XIA Jieying¹, HONG Yang¹, DONG Han¹, LIU Yanglu¹, ZHOU Jing¹, YUAN Mingming¹, WANG Yongjin¹, YAN Liangchun¹^,^*()

1. Center of Experimental Animals, Sichuan Academy of Chinese Medicine Sciences, Chengdu 610041, China
2. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China

Received:2021-12-22 Online:2022-08-25 Published:2022-08-26
Contact: YAN Liangchun

摘要/Abstract

摘要：

为了探究天然化合物毛蕊花糖苷(verbascoside, VBS)对2型猪链球菌(Streptococcus suis serotype 2, SS2)致病性的作用,本研究通过测定最低抑菌浓度、绘制细菌生长曲线及红细胞溶血试验,发现低浓度下的毛蕊花糖苷不影响猪链球菌的正常生长,但对其溶血活性有显著抑制作用;免疫印迹试验表明,毛蕊花糖苷不影响2型猪链球菌分泌溶血素蛋白(suilysin, SLY);通过分子对接预测毛蕊花糖苷可与SLY的2个氨基酸残基(SER-388、GLN-441)形成3条氢键结合从而使毛蕊花糖苷嵌入SLY蛋白的第4结构域,同时体外溶血活性试验进一步证实毛蕊花糖苷可直接抑制SLY介导的溶血活性;最后,小鼠体内试验结果表明,毛蕊花糖苷可明显提高2型猪链球菌感染小鼠的存活率。综上,毛蕊花糖苷在不影响2型猪链球菌生长活性的情况下,可通过抑制溶血素活性来降低2型猪链球菌对小鼠的致病性。

关键词: 2型猪链球菌, 溶血素, 毛蕊花糖苷, 致病性

Abstract:

The aim of this study is to explore the effect of verbascoside (VBS), a natural compound, on the pathogenicity of Streptococcus suis serotype 2 (SS2). By determining the minimum inhibitory concentration (MIC), growth curve of SS2 and hemolysis activity of erythrocytes, is was found that the low concentration of VBS significantly inhibited the hemolytic activity of SS2 without interfering with its growth. Western blotting showed that VBS had no influence on the expression of suilysin (SLY) in SS2. It was predicted by molecular docking that VBS could be embedded into the domain 4 of SLY, which was through the formation of three hydrogen bonds between VBS and two residues (SER-388, GLN-441) of SLY. And it was further confirmed that VBS directly inhibited the hemolytic activity of SLY in vitro. Moreover, VBS significantly increased the survival rate of mice infected with SS2 in vivo. In conclusion, without affecting the growth of SS2, VBS can inhibit the hemolytic activity of SLY to reduce the pathogenicity of SS2 in mice.

Key words: Streptococcus suis serotype 2, suilysin, verbascoside, pathogenicity

中图分类号:

S858.28

詹佳飞, 徐魁, 张磊, 夏介英, 洪杨, 董涵, 刘洋露, 周静, 袁明铭, 王永金, 鄢良春. 毛蕊花糖苷抑制2型猪链球菌的溶血素蛋白活性而降低其小鼠致病性[J]. 浙江农业学报, 2022, 34(8): 1609-1616.

ZHAN Jiafei, XU Kui, ZHANG Lei, XIA Jieying, HONG Yang, DONG Han, LIU Yanglu, ZHOU Jing, YUAN Mingming, WANG Yongjin, YAN Liangchun. Verbascoside lowers Streptococcus suis serotype 2 pathogenicity in mice by inhibiting hemolytic activity of suilysin[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1609-1616.

图/表 7

图1 在不同浓度毛蕊花糖苷作用下2型猪链球菌的生长曲线

Fig.1 Growth curves of SS2 treated with different concentrations of VBS

图2 毛蕊花糖苷与2型猪链球菌共同培养体系中上清的溶血活性 NC, 阴性对照。*表示与未添加毛蕊花糖苷组差异显著 (P<0.05),**表示与未添加毛蕊花糖苷组差异极显著 (P<0.01)。下同。

Fig.2 Hemolytic activity in supernatant of VBS and SS2 co-culture system NC, Negative control. * meaned significant difference compared with VBS-free group (P<0.05), ** meaned extremely significant difference compared with VBS-free group (P<0.01). The same as below.

图3 毛蕊花糖苷与2型猪链球菌共同培养体系中上清的SLY分泌量

Fig.3 Expression of SLY in supernatant of VBS and SS2 co-culture system

图4 毛蕊花糖苷与SLY蛋白的分子对接构象 A,毛蕊花糖苷二维化学结构;B,毛蕊花糖苷三维化学结构;C, 毛蕊花糖苷的羟基与SLY残基结合细节。

Fig.4 The molecular docking conformation of VBS with SLY A, 2D chemical structure of VBS; B, 3D chemical structure of VBS; C, Details of binding between the hydroxyl group of VBS and the residues of SLY.

图5 SLY重组蛋白的表达及纯化 M, 蛋白质分子量标准;1,加IPTG诱导大肠埃希菌BL21表达SLY重组蛋白;2,纯化后的SLY重组蛋白。

Fig.5 Expression and purification of recombinant SLY M, Protein molecular weight marker; 1, Lysate of IPTG-induced E.coil BL21 expressing recombinant SLY; 2, Purified recombinant SLY.

图6 毛蕊花糖苷对纯化后SLY溶血活性的作用

Fig.6 Effect of VBS on the hemolytic activity of purified SLY

图7 毛蕊花糖苷对2型猪链球菌感染小鼠存活率的影响

Fig.7 Effect of VBS on the survival rate of mice infected with SS2

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毛蕊花糖苷抑制2型猪链球菌的溶血素蛋白活性而降低其小鼠致病性

Verbascoside lowers Streptococcus suis serotype 2 pathogenicity in mice by inhibiting hemolytic activity of suilysin

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