Abstract
The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.
Highlights
The amoebae-resistant environmental bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires’ disease [1,2]
Legionella pneumophila is an environmental bacterium that grows within free-living amoebae and, upon inhalation, in human lung macrophages, causing the severe pneumonia Legionnaires’ disease
L. pneumophila injects via a dedicated secretion apparatus about 300 different ‘‘effector’’ proteins directly into host cells, where they interfere with cellular processes
Summary
The amoebae-resistant environmental bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires’ disease [1,2]. In free-living amoebae as well as in macrophages of the innate immune system, L. pneumophila employs an apparently conserved mechanism to form a replication-permissive membrane-bound compartment, the ‘‘Legionellacontaining vacuole’’ (LCV) [3,4,5]. Microtubules play a role in the initial trafficking events of LCVs, prior to the acquisition of the early secretory vesicle marker GFP-HDEL and the resident ER marker calnexin-GFP [6]. LCVs revealed more than 560 host proteins, including a- and btubulin, as well as a number of small GTPases and GTPaseinteracting factors [7]. In addition to Arf and Rab GTPases implicated in the secretory and endosomal vesicle trafficking pathways, Ran and its effector Ran binding protein 1 (RanBP1) were identified in this study as LCV host components
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