Department of Microbiology and Immunology, College of Medicine, University of Louisville, Kentucky, USA.
PLoS Pathog. 2009 Dec;5(12):e1000704. doi: 10.1371/journal.ppat.1000704. Epub 2009 Dec 24.
The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.
嗜肺军团菌在水生环境中和巨噬细胞内各种原生动物中增殖的能力表明，它具有非凡的进化能力，并巧妙地利用了进化上保守的真核生物过程。嗜肺军团菌的锚蛋白 B (AnkB) 是一种非典型的含 F-box 蛋白，是唯一已知的 Dot/Icm 易位效应物，对于在巨噬细胞和原生动物宿主的空泡内进行腔内增殖是必需的。我们表明，AnkB 的 F-box 结构域和（9）L(10)P 保守残基对于细菌的细胞内增殖以及在巨噬细胞、Dictyostelium discoideum 和 Acanthamoeba 中快速获得含有军团菌的空泡（LCV）的多泛素化蛋白是必需的。有趣的是，在细菌附着后 5 分钟内，细胞外细菌迅速触发 AnkB 的易位和多泛素化蛋白在巨噬细胞和 Acanthamoeba 中的募集。在哺乳动物细胞中异位表达的 AnkB 定位于细胞的外周，与宿主 SKP1 共定位，并募集多泛素化蛋白，这导致 ankB 突变体的细胞内生长恢复到类似于亲本菌株的水平。虽然异位表达的 AnkB-(9)L(10)P/AA 变体定位于细胞外周，但它不能募集多泛素化蛋白，并且不能挽救 ankB 突变体的细胞内生长缺陷。已经证明 AnkB 与宿主 SKP1 之间存在直接的体内相互作用，但 AnkB-(9)L(10)P/AA 变体则不然。重要的是，SKP1 的 RNAi 介导的表达沉默使细胞对嗜肺军团菌的细胞内增殖变得不允许。AnkB 在利用多泛素化机制方面的作用对于肺炎军团菌在小鼠军团病模型中的肺部增殖是必需的。因此，AnkB 表现出真核 F-box 蛋白的新型分子和功能模拟，利用保守的多泛素化机制在进化上遥远的宿主中进行细胞内增殖。
