Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection
(A) Ran accumulates on LCVs. D. discoideum producing RanA-GFP was infected (MOI 50, 1 h) with DsRed-producing L. pneumophila wild-type, ΔlegG1 or ΔicmT harboring pSW001 and immuno-stained for the LCV membrane marker SidC. LCVs in lysates of infected cells are shown. (B) Depletion of Ran or RanBP1 inhibits intracellular growth of L. pneumophila. A549 lung epithelial carcinoma cells were treated with AllStars siRNA (negative control) or with siRNA oligonucleotides targeting Ran, RanBP1 or Arf1 (positive control) for 2 d, and intracellular replication of GFP-producing L. pneumophila harboring pNT28 was quantified by fluorescence measurements after 24 h. Data represent mean and standard deviation of three independent experiments considering the 3 most effective out of 4 different oligonucleotides (Fig. S1B). Student's t-test; ***, p<0.001. (C) Icm/Dot-dependent localization of M45-LegG1 on LCVs in cell homogenates. D. discoideum producing calnexin-GFP was infected (MOI 50) with L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pSU19 (M45-LegG1) or with ΔlegG1 harboring pCR033 (vector), homogenized and immuno-stained with an anti-M45 antibody and with DAPI. The percentage of M45-LegG1-positive LCVs (n = 100/strain, 3 independent experiments) was scored in lysates of infected cells (*, p<0.05). Bars (A, C), 0.5 µm.