Chemical synthesis of LAI-1 and amino-LAI-1.

<p>The chemical synthesis of the putative <i>L</i>. <i>pneumophila</i> small molecule compounds (A) LAI-1 and (B) amino-LAI-1 is shown.</p

LAI-1-dependent inhibition of cell migration requires the Cdc42 GEF ARHGEF9.

<p>(A) Confluent cell layers of A549 cells were treated for 2 days with siRNA against the different Cdc42 GEFs or GAPs indicated. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified at 6 different positions per condition using ImageJ software. Means and standard deviations of 3 samples are shown, which are representative of 3 independent experiments (***<i>p</i> < 0.001).</p

LAI-1 reverses Icm/Dot-dependent inhibition of migration by <i>L</i>. <i>pneumophila</i>.

<p>(A) <i>D</i>. <i>discoideum</i> Ax3 amoebae harboring pSW102 (GFP) or (B) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria and treated with different concentrations of LAI-1 (1, 5 and 10 μM) or not. The effect of LAI-1 on migration of amoebae towards folate (1 mM) or macrophages towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 hours. Macrophages were stained with Cell Tracker Green BODIPY. Graphs depict the per cent fluorescence intensity versus migration distance. (C) <i>D</i>. <i>discoideum</i> Ax3 amoebae harboring pSW102 (GFP) or (D) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria and treated with LAI-1 (10 μM, 1 h) or not. Single cell migration towards folate (1 mM) or CCL5 (100 ng/ml) was tracked in an under-agarose assay for 15 min or 1 h, respectively. Motility parameters (forward migration index, FMI, and velocity (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005307#ppat.1005307.s007" target="_blank">S7 Fig</a>)) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria, treated with LAI-1 (10 μM) or not, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (F) The scratch area was quantified at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (C, D, F; means and standard deviations; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001).</p

Dose-dependent inhibition of chemotaxis and cell migration by LAI-1.

<p><i>D</i>. <i>discoideum</i> amoebae harboring pSW102 (GFP) were treated for 1 h with different concentrations of (A) racemic LAI-1, (B) 10 μM (<i>R</i>)-LAI-1, (<i>S</i>)-LAI-1, (<i>R</i>)-amino-LAI-1 or (<i>S</i>)-amino-LAI-1, or (C) different concentrations of CAI-1, and cell migration towards folate (1 mM) was monitored in under-agarose assays for 4 h. Graphs depict per cent GFP fluorescence intensity versus migration distance. (D) <i>D</i>. <i>discoideum</i> amoebae harboring pSW102 (GFP) were treated with LAI-1 (10 μM, 1 h). Single cell migration towards folate (1 mM) was monitored in under-agarose assays for 15 min. Motility parameters (forward migration index, FMI; and velocity) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Murine RAW 264.7 macrophages were treated for 1 h with different concentrations of racemic LAI-1, cell migration towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 h, and the cells were stained with Cell Tracker Green BODIPY. Macrophages treated for 1 h with 10 μM LAI-1 were immuno-labeled for (F) α-tubulin (green) or (G) actin (red) and, as a control, the production of cellular α-tubulin or actin was quantified by Western blot. Microtubule fibers per cell were counted along cross-sections (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005307#ppat.1005307.s003" target="_blank">S3 Fig</a>), and the actin architecture was analyzed by quantifying the number of cells displaying cortical actin. The graphs show means and standard deviations of 3 independent experiments (n > 25 (α-tubulin) or > 40 (actin) single cells; Student´s t-test, *<i>p</i> < 0.05, **<i>p</i> < 0.01). Bars (F, G), 5 μm.</p

LAI-1-dependent inhibition of cell migration requires IQGAP1 and Cdc42.

<p>(A) Confluent cell layers of A549 epithelial cells were treated with siRNA against IQGAP1, Cdc42, RhoA or Rac1 for 2 days. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of triplicate samples are shown (**<i>p</i> < 0.01). The data is representative of 3 independent experiments.</p

Effect of <i>L</i>. <i>pneumophila lqs</i> genes on host cell migration.

<p><i>D</i>. <i>discoideum</i> strain Ax3 producing GFP (pSW102) was infected (MOI 10, 1 h) with (A) <i>L</i>. <i>pneumophila</i> wild-type, Δ<i>icmT</i>, Δ<i>lqsS</i>, Δ<i>lqsT</i>, <i>ΔlqsS-lqsT</i>, Δ<i>lqsR</i> or Δ<i>lqsA</i> mutant strains harboring pSW001 (DsRed), or with (D) the strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA). An under-agarose assay was used to monitor the migration towards folate (1 mM) for another 4 h. The white lines represent the edge of the sample wells. (B, E) Graphs of the data from (A, D) plotted as per cent GFP fluorescence intensity versus migration distance. (C) Murine RAWs 264.7 macrophages were infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type, Δ<i>icmT</i>, Δ<i>lqsS</i>, Δ<i>lqsT</i>, <i>ΔlqsS-lqsT</i>, Δ<i>lqsR</i> or Δ<i>lqsA</i> mutant strains. Cells were stained with Cell Tracker Green BODIPY and let migrate towards CCL5 (100 ng/ml) in an under-agarose assay for another 4 h. Graphs show the per cent fluorescence intensity versus migration distance. (F) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>lqsA</i> mutant strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (G) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of the triplicate samples are shown (pNT28 vs. pNT36: ***<i>p</i> < 0.001). The data shown are representative of at least 3 independent experiments.</p

Model of LA1-1-dependent inter-kingdom signaling through IQGAP1, Cdc42 and ARHGEF9.

<p>In eukaryotic cells, LAI-1 (directly or indirectly) inhibits or prevents the activation of the Cdc42-specific GEF ARHGEF9, which in turn prevents the IQGAP1-dependent activation of Cdc42. The host cell might detect extracellular and/or intracellular LAI-1.</p

Migration inhibition by <i>L</i>. <i>pneumophila</i> is augmented in the absence of Cdc42.

<p>(A) Confluent cell layers of A549 cells were treated with (A) scrambled siRNA or siRNA against (B) Cdc42 or (C) Rac1 for 2 days, left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified after 24 h at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (***<i>p</i> < 0.001). (C) <i>L</i>. <i>pneumophila</i> colocalizes with IQGAP1 and Cdc42. A549 cells were infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria harboring plasmid pSW001 (DsRed), and the subcellular localization of the scaffold protein (green; FITC) or the small GTPase (green; FITC) was analyzed by confocal microscopy using antibodies against IQGAP1 or Cdc42. Nuclei were stained with DAPI (blue). Bars, 5 μm.</p

LAI-1 promotes inactivation of Cdc42 and redistribution of IQGAP1 to the cell cortex.

<p>(A) A549 cells were treated with LAI-1 (10 μM, 1 h), and the activation state of Cdc42 was analyzed by Western blot using an antibody recognizing Cdc42(GTP/GDP) (left panel). Quantification by densitometry was performed using ImageJ (right panel). A549 cells were treated with LAI-1 (10 μM, 1 h), fixed, stained with antibodies against (B) IQGAP1 or (C) Cdc42 and analyzed by confocal microscopy (left panels; green, FITC; blue, DAPI). The graphs (right panels) are based on the relative fluorescence intensity along cell sections (n = 50, ***<i>p</i> < 0.001) and show that upon LAI-1 treatment IQGAP1 but not Cdc42 redistributes to the cell cortex. Bars (B, C), 5 μm.</p