LegG1 promotes intracellular replication of <i>L. pneumophila</i>.

<p>(<b>A</b>) Intracellular replication in RAW264.7 macrophages infected (MOI 0.1) with <i>L. pneumophila</i> wild-type strain JR32, Δ<i>icmT</i> or Δ<i>legG1</i>. The infected cells were lysed and CFU were determined. Data shows means and standard deviations of triplicates and is representative of three independent experiments (*, <i>p</i><0.05; ***, <i>p</i><0.001). (<b>B</b>) For competition assays <i>A. castellanii</i> amoebae were co-infected with wild-type <i>L. pneumophila</i> and the Δ<i>legG1</i> 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 <i>L. pneumophila</i> wild-type, Δ<i>icmT</i>, or Δ<i>legG1</i> harboring pCR033, or with Δ<i>legG1</i>/pSU19 (M45-LegG1), and (<b>C</b>) analyzed by transmission electron microscopy (TEM) after embedding the sample in epoxy resin, or (<b>D</b>) analyzed by flow cytometry after detaching the cells by scraping and staining with the membrane integrity marker propidium iodide (1 µg/ml). (<b>E</b>) For TEM analysis of Golgi stacks, RAW264.7 macrophages were infected with <i>L. pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>legG1</i> (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. (<b>F</b>) Single round replication assay in RAW264.7 macrophages infected (MOI 20) with GFP-producing <i>L. pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>legG1</i> 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.