SILAC and LC-MS/MS identification of Streptococcus equi ssp. zooepidemicus proteins that contribute to mouse brain microvascular endothelial cell infection
SILAC and LC-MS/MS identification of Streptococcus equi ssp. zooepidemicus proteins that contribute to mouse brain microvascular endothelial cell infection
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DOI：
10.1007/s00253-016-7579-4
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发表时间：
2016-05
影响因子：
5
通讯作者：
Ma Zhe;Peng Jie;Z. Hui;Xu Bin;Xiaomeng Pei;Huixing Lin;Chengping Lu;Hongjie Fan
中科院分区：
文献类型：
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作者：
Ma Zhe;Peng Jie;Z. Hui;Xu Bin;Xiaomeng Pei;Huixing Lin;Chengping Lu;Hongjie Fan
马链球菌兽疫亚种（SEZ）在人和动物中引起脑膜炎。SEZ的一些解离蛋白对小鼠脑微血管内皮细胞（mBMEC）具有细胞毒性，并可能有助于SEZ穿过血脑屏障（BBB）。在本研究中，证实了SEZ穿透体外BBB模型的能力。我们使用细胞培养物中氨基酸的稳定同位素标记（SILAC）来标记具有重或轻同位素标记的氨基酸的SEZ蛋白，沿着LC-MS/MS来确定哪些SEZ蛋白参与与mBMEC的相互作用。SEZ蛋白质同位素标记效率为94.7%，足以用于进一步分析。49个标记的肽被鉴定为与mBMEC结合，其与25个SEZ蛋白相匹配。生物信息学分析表明，这些蛋白质主要分布在细胞质中。这些蛋白质可能具有破坏宿主血脑屏障的功能，其中一些蛋白质是其他细菌中已知的毒力因子。间接免疫荧光结果表明SEZ烯醇化酶对mBMEC具有结合活性。保护性试验结果表明，烯醇化酶是抗SEZ感染的保护性抗原。这项研究是SILAC与LC-MS/MS相结合的首次应用，以鉴定可能有助于mBMEC感染的SEZ蛋白，并可能显示与破坏BBB相关的功能。这些结果为研究SEZ诱导的脑膜炎的机制提供了许多未来的途径。
Streptococcus equissp.zooepidemicus(SEZ) causes meningitis in both humans and animals. Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells (mBMECs) and may contribute to the penetration of SEZ across the blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged amino acids, along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs. The efficiency of SEZ protein isotope labeling was 94.7 %, which was sufficient for further analysis. Forty-nine labeled peptides were identified as binding to mBMECs, which matched to 25 SEZ proteins. Bioinformatic analysis indicated that most of these proteins were cytoplasmic. These proteins may have functions in breaching the host BBB, and some of them are known virulence factors in other bacteria. Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs. Protective test results showed that enolase was a protective antigen against SEZ infection. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis.