Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion
Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion
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DOI：
10.1046/j.1365-2958.2000.02027.x
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发表时间：
2000-08-01
影响因子：
3.6
通讯作者：
Koronakis, V
中科院分区：
文献类型：
--
作者：
Hayward, RD;McGhie, EJ;Koronakis, V
沙门氏菌感染的早期事件是非吞噬性肠上皮细胞的入侵。病原体被巨噬细胞吞噬，这是由细菌蛋白的接触依赖性传递引起的，这种传递会破坏信号通路并促进细胞骨架重排。SipB是沙门氏菌传递和侵袭所需的一种蛋白，被证明可以定位于入侵哺乳动物靶细胞的细菌的细胞表面，并与外膜蛋白分离。为了研究SipB的性质，我们在重组大肠杆菌中表达后纯化了天然全长蛋白。纯化的SipB通过n端蛋白酶抗性结构域组装成六聚体，预测形成三聚体卷曲线圈，使人想起指导同型膜融合的病毒包膜蛋白。SipB蛋白既能整合到哺乳动物细胞膜上，也能整合到磷脂囊泡中，而不破坏双层的完整性，并能诱导脂质体融合，这种融合在中性pH下最理想，且受膜脂组成的影响。SipB引导异型融合，允许大肠杆菌衍生脂质体的内容物进入活哺乳动物细胞的细胞质。
An early event in Salmonella infection is the invasion of non-phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact-dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full-length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N-terminal protease-resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli-derived liposomes into the cytosol of living mammalian cells.