Hume Peter J, McGhie Emma J, Hayward Richard D, Koronakis Vassilis
University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, UK.
Mol Microbiol. 2003 Jul;49(2):425-39. doi: 10.1046/j.1365-2958.2003.03559.x.
An essential early event in Shigella and Salmonella pathogenesis is invasion of non-phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full-length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N-terminal domain predicted to form a trimeric coiled-coil, and is predominantly alpha-helical. Upon lipid interaction, two transmembrane domains (residues 313-333 and 399-419) penetrate the bilayer, allowing the intervening hydrophilic region (334-398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB-derived 162 residue alpha-helical polypeptide (IpaB(418-580)) is a potent inhibitor of IpaB-directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10(-8) M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB(418-580) failed to prevent the contact-dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact-dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.
志贺氏菌和沙门氏菌发病机制中的一个重要早期事件是侵袭非吞噬性肠道上皮细胞。病原体进入是由多种细菌效应蛋白传递到靶哺乳动物细胞中引发的。插入宿主质膜的志贺氏菌侵袭质粒抗原B（IpaB）是效应蛋白传递和侵袭所必需的。为了研究IpaB的生化特性和膜拓扑结构，我们在实验室大肠杆菌中表达后纯化了天然全长蛋白。纯化的IpaB通过预测形成三聚体卷曲螺旋的N端结构域组装成三聚体，并且主要是α螺旋结构。与脂质相互作用时，两个跨膜结构域（残基313 - 333和399 - 419）穿透双层膜，使中间的亲水区（334 - 398）穿过膜。纯化的IpaB整合到模型膜、红细胞膜和哺乳动物细胞膜中而不破坏双层膜的完整性，并在体外诱导脂质体融合。源自IpaB的162个残基的α螺旋多肽（IpaB(418 - 580)）在体外是IpaB介导的脂质体融合的有效抑制剂，并且在10^(-8) M时阻断志贺氏菌进入培养的哺乳动物细胞。它也是沙门氏菌侵袭蛋白B（SipB）活性和沙门氏菌进入的异源抑制剂。相反，IpaB(418 - 580)未能阻止志贺氏菌的接触依赖性溶血活性。这些发现对所提出的接触依赖性溶血与志贺氏菌进入之间的直接联系提出了质疑，并表明IpaB和SipB具有共同的生化特性和膜拓扑结构，这与细胞进入过程中保守的作用模式一致。
