Activation of Ran GTPase by a Legionella effector promotes microtubule polymerization, pathogen vacuole motility and infection

Autor: Gudrun Pfaffinger, Heinrich Grabmayr, Eva Rothmeier, Aymelt Itzen, Stefan Wölke, Mandy Hannemann, Gareth Griffiths, Hubert Hilbi, Andreas R. Bausch, Urska Repnik, Christopher F. Harrison, Annette Müller-Taubenberger, Christine Hoffmann
Rok vydání: 2013
Předmět:
Zdroj: PLoS Pathogens
PLoS Pathogens, Vol 9, Iss 9, p e1003598 (2013)
ISSN: 1553-7374
Popis: 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.
Author Summary Legionella pneumophila is an environmental bacterium that grows within free-living amoebae and, upon inhalation, in human lung macrophages, thus causing the severe pneumonia Legionnaires' disease. Within amoebae or macrophages the bacteria form a distinct membrane-bound replication niche, the “Legionella-containing vacuole” (LCV). To this end, L. pneumophila injects via a dedicated secretion apparatus about 300 different “effector” proteins directly into host cells, where they interfere with cellular processes. LCV formation is poorly understood, and the function and targets of most bacterial effector proteins are unknown. In this study, we characterize an L. pneumophila effector protein that activates the small host GTPase Ran, which is essential for crucial cellular processes, such as spindle assembly and cytokinesis, nucleo-cytoplasmic transport, as well as nuclear envelope formation. We discovered that Ran promotes intracellular replication of L. pneumophila and its activation on the LCV membrane by LegG1 causes the polymerization of microtubules, along which cellular vesicles as well as LCVs move within cells. Our study defines a novel strategy how pathogenic bacteria subvert host processes to promote intracellular survival and replication.
Databáze: OpenAIRE
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