重组耶尔森氏假结核菌侵袭蛋白细胞内行为的观察

Observation of the intracellular behavior of recombinant Yersinia pseudotuberculosis invasin protein.

作者信息

Koga Hisashi, Izawa Ichiro, Araki Norie, Saya Hideyuki, Mimori Tatsuyuki

机构信息

Department of Tumor Genetics and Biology, Graduate School of Medical Science Kumamoto University, Kumamoto, Japan.

出版信息

Microbiol Immunol. 2005;49(4):297-302. doi: 10.1111/j.1348-0421.2005.tb03729.x.

DOI:10.1111/j.1348-0421.2005.tb03729.x

PMID:15840954

Abstract

In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, (125) I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.

摘要

在本研究中，我们观察了重组侵袭素（一种103 kDa的假结核耶尔森菌外膜蛋白）的细胞内行为。为模拟细菌侵袭素的体内行为，通过将生物素化的谷胱甘肽S-转移酶（GST）融合侵袭素C端部分蛋白（GST-INVS）与抗生物素蛋白孵育，生成了一种多价形式的侵袭素。多项实验证实，重组侵袭素能够持续重现侵袭素介导的进入哺乳动物上皮细胞的过程。我们通过蛋白质免疫印迹法、¹²⁵I摄取和免疫荧光显微镜分析了多价侵袭素的分子动力学。内化的多价侵袭素迅速转移至RIPA不溶性（富含聚合肌动蛋白）组分并形成细胞质囊泡，而单价侵袭素则未表现出这种动力学。基于这些观察结果，我们得出结论，我们的无细菌系统能够分析侵袭素在假结核耶尔森菌进入过程中的作用。