Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles
Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles
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DOI：
10.1371/journal.pone.0150166
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发表时间：
2016-03-25
期刊：
影响因子：
3.7
通讯作者：
Chromy, Brett A.
中科院分区：
文献类型：
--
作者：
Coleman, Matthew A.;Cappuccio, Jenny A.;Chromy, Brett A.
鼠疫耶尔森氏菌进入宿主细胞并逃避宿主防御，部分是通过鼠疫耶尔森氏菌蛋白和宿主膜之间的相互作用。其中一种相互作用是通过III型分泌系统，该系统对鼠疫杆菌外膜效应蛋白转位使用高度保守和有序的复合体，称为注射体。注射体中直接与宿主细胞膜相互作用的部分被称为转位子。转位子被认为形成了一个孔，允许效应器分子进入宿主细胞。为了便于对转运子机制的研究，我们开发了一种无细胞方法来表达转运子孔蛋白作为双层膜模拟纳米支架(NLP)中的复合体。初步结果表明，在脂质体的存在下，鼠疫菌外膜蛋白YopB和YopD的无细胞表达得到增强。然而，这些络合物倾向于聚集和沉淀。随着共表达(NLP)形成组分的加入，YopB和/或YopD复合体变得可溶，增加了用于生物物理研究的蛋白质产量。用原子力显微镜和荧光相关光谱等生物物理方法证实了可溶性YopB/D复合体与NLP的结合。免疫沉淀法证实YopB/D复合体与NLP之间存在相互作用。YopB/D转位蛋白复合体嵌入在NLP中，为研究病原菌和宿主蛋白之间的蛋白质相互作用提供了一个平台。这些研究将有助于阐明这种病原体能够将效应蛋白注入宿主细胞从而逃避宿主防御的鲜为人知的机制。
Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.