Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

PLoS Pathogens

Aymelt Itzen¹  Mandy Hannemann¹  Urska Repnik²  Gareth Griffiths²  Annette Müller-Taubenberger³  Heinrich Grabmayr⁴  Andreas Bausch⁴  Christopher F. Harrison⁵  Eva Rothmeier⁵  Christine Hoffmann⁵  Hubert Hilbi⁵  Gudrun Pfaffinger⁵  Stefan Wölke⁵ 

[1] Center for Integrated Protein Science Munich, Department of Chemistry, Technische Universität München, Garching, Germany;Department of Molecular Biosciences, University of Oslo, Oslo, Norway;Institute for Anatomy and Cell Biology, Department of Medicine, Ludwig-Maximilians Universität München, München, Germany;Institute of Molecular and Cellular Biophysics, Department of Physics, Technische Universität München, Garching, Germany;Max von Pettenkofer-Institute, Department of Medicine, Ludwig-Maximilians Universität München, München, Germany

关键词: Legionella pneumophila;    Macrophages;    Microtubules;    Dictyostelium discoideum;    Yersinia enterocolitica;    Amoebas;    Guanine nucleotide exchange factors;    Guanosine triphosphatase;   

DOI  :  10.1371/journal.ppat.1003598

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY