Pistor S, Chakraborty T, Walter U, Wehland J
Gesellschaft für Biotechnologische Forschung, Abteilung Zellbiologie und Immunologie, Braunschweig, Germany.
Curr Biol. 1995 May 1;5(5):517-25. doi: 10.1016/s0960-9822(95)00104-7.
Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system.
We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP.
Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.
包括单核细胞增生李斯特菌在内的几种细胞内病原体利用宿主基于肌动蛋白的细胞骨架成分进行细胞内移动和细胞间传播。这些细菌系统提供了相对简单的模型系统，用于研究基于肌动蛋白的运动性。对单核细胞增生李斯特菌的遗传分析导致鉴定出90 kD表面结合的ActA多肽是启动宿主肌动蛋白丝募集所需的唯一细菌因子。一旦单核细胞增生李斯特菌进入哺乳动物细胞，许多宿主肌动蛋白结合蛋白就会定位在围绕它的基于肌动蛋白的细胞骨架内，包括α-辅肌动蛋白、丝束蛋白、细丝蛋白、绒毛蛋白、埃兹蛋白/根蛋白、前纤维蛋白和血管舒张刺激磷蛋白VASP。已知只有VASP直接与ActA结合。我们试图确定ActA分子的哪些区域与VASP和宿主微丝系统的其他成分相互作用。
我们使用先前开发的线粒体靶向测定法来确定ActA蛋白中参与募集宿主基于肌动蛋白的细胞骨架的区域。通过检查氨基末端截短的ActA衍生物募集细胞骨架蛋白的能力，在ActA的第128至151位氨基酸之间鉴定出肌动蛋白丝成核的必需元件。一个缺失中央富含脯氨酸重复序列的ActA衍生物保留了募集丝状肌动蛋白的能力，尽管能力较弱，但无法结合VASP。
我们的研究揭示了入侵的李斯特菌与宿主微丝蛋白之间发生的初始相互作用。李斯特菌ActA多肽包含至少两个有效微丝组装所需的必需位点：一个用于肌动蛋白丝成核的氨基末端23个氨基酸区域，以及VASP结合的富含脯氨酸重复序列。因此，ActA代表了一种原型肌动蛋白丝成核剂。我们认为存在ActA的宿主细胞类似物，并且它们是参与细胞运动的结构的重要组成部分。
