Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection
(A) Intracellular replication in RAW264.7 macrophages infected (MOI 0.1) with L. pneumophila wild-type strain JR32, ΔicmT or ΔlegG1. The infected cells were lysed and CFU were determined. Data shows means and standard deviations of triplicates and is representative of three independent experiments (*, p<0.05; ***, p<0.001). (B) For competition assays A. castellanii amoebae were co-infected with wild-type L. pneumophila and the ΔlegG1 mutant strain at a 1∶1 ratio (MOI of 0.01 each) and grown at 37°C during 21 d. Every third day the supernatant and lysed amoebae were diluted 1∶5000, fresh amoebae were infected, and CFU determined on CYE agar plates containing kanamycin or not. The data shown are means and standard deviations of triplicates and representative of 3 independent experiments. For toxicity assays RAW264.7 macrophages were infected (MOI 10, 4 h) with L. pneumophila wild-type, ΔicmT, or ΔlegG1 harboring pCR033, or with ΔlegG1/pSU19 (M45-LegG1), and (C) analyzed by transmission electron microscopy (TEM) after embedding the sample in epoxy resin, or (D) analyzed by flow cytometry after detaching the cells by scraping and staining with the membrane integrity marker propidium iodide (1 µg/ml). (E) For TEM analysis of Golgi stacks, RAW264.7 macrophages were infected with L. pneumophila wild-type, ΔicmT or ΔlegG1 (MOI 10) and embedded in epoxy resin. The total length of Golgi cisternae and the ratio of the total number of Golgi cisternae relative to the total cytoplasmic area were quantified by stereology in thin sections. Bars, 1 µm. (F) Single round replication assay in RAW264.7 macrophages infected (MOI 20) with GFP-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pNT28, in presence or absence of 10 µM nocodazole. Means and standard deviations of 3 samples per strain, each analyzed in triplicate, from a single experiment is shown. Data are representative of 3 independent experiments.