Purification and characterization of the subtilisin-like protease of Streptococcus suis that contributes to its virulence
Purification and characterization of the subtilisin-like protease of Streptococcus suis that contributes to its virulence
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DOI：
10.1016/j.vetmic.2010.09.024
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发表时间：
2011-03-24
影响因子：
3.3
通讯作者：
Grenier, Daniel
中科院分区：
文献类型：
--
作者：
Bonifait, Laetitia;Vaillancourt, Katy;Grenier, Daniel
猪链球菌是一种主要的猪病原体，可引起脑膜炎、心内膜炎和败血症等严重感染。猪链球菌也被认为是一种人畜共患病病原体，并表达多种毒力因子。最近发现的猪链球菌类枯草杆菌蛋白水解酶(SSPA)在动物模型中对该细菌的致病性起着重要作用。本研究的目的是克隆、纯化和鉴定猪链球菌P1/7型的SSPA基因。将其克隆到原核表达载体pBAD/hisB中，并将其插入到大肠杆菌中进行表达。重组酶经层析纯化，经SDS-PAGE分析，其相对分子质量为170 kDa。该酶的最适pH为7，最适温度为25℃~37℃，对发色底物琥珀酰丙氨酸-丙氨酸-Pro-苯丙氨酸-PNA具有较高的专一性，丝氨酸蛋白酶的特异性抑制剂则抑制其活性。除了降解明胶外，该酶还能降解纤维蛋白原的Aα链，从而阻止凝血酶形成纤维蛋白。重组类枯草杆菌蛋白水解酶对脑微血管内皮细胞也有毒性作用。最后，感染猪链球菌的猪血清与重组SSPA发生反应，表明重组SSPA是在感染过程中产生的。综上所述，猪链球菌的SSPA与其他致病链球菌产生的类枯草杆菌蛋白水解酶具有相似性，可能参与了猪链球菌感染的致病过程。(C)2010爱思唯尔B.V.保留所有权利。
Streptococcus suis is a major swine pathogen that is responsible for severe infections such as meningitis, endocarditis, and septicemia. S. suis is also recognized as a zoonotic agent and expresses several virulence factors. The recently identified subtilisin-like protease (SspA) of S. suis plays an important role in the pathogenicity of this bacterium in animal models. The objective of the present study was to clone, purify, and characterize the SspA of serotype 2 S. suis P1/7. The SSU0757 gene encoding SspA was amplified and a 4798-bp DNA fragment was obtained. It was cloned into the expression plasmid pBAD/HisB and then inserted into Escherichia coli to overproduce the protein. The recombinant protease was purified by chromatography procedures and showed a molecular weight of 170 kDa by SDS-PAGE. Its activity was optimal at pH 7 and at temperatures ranging from 25 degrees C to 37 degrees C. It had a high specificity for the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-pNa while specific inhibitors of serine proteases inhibited its activity. In addition to degrading gelatin, the protease hydrolyzed the A alpha chain of fibrinogen, which prevented fibrin formation by thrombin. The recombinant subtilisin-like protease also showed toxicity towards brain microvascular endothelial cells. Lastly, sera from pigs infected with S. suis reacted with the recombinant SspA, indicating that it is produced during infections. In conclusion, the SspA of S. suis shared similarities with subtilisin-like proteases produced by other pathogenic streptococci and may contribute to the pathogenic process of S. suis infections. (C) 2010 Elsevier B.V. All rights reserved.