A Role for PI3Ks in Degradation of L. pneumophila Δ<i>icmT</i>

<p>Quantification by flow cytometry of intracellular degradation of GFP-labeled L. pneumophila Δ<i>icmT</i> (MOI 100, MOI 500) within wild-type <i>Dictyostelium</i> or Δ<i>PI3K1/2.</i> In the absence of PI3Ks, Δ<i>icmT</i> is less efficiently phagocytosed and degraded.</p

Binding of L. pneumophila Icm/Dot–Secreted Proteins to PIs In Vitro

<div><p>(A) Binding of affinity-purified GST fusion proteins of SidC, SdcA, SidD, or SdhB (160 pmol) to different lipids (100 pmol; left panels) or 2-fold serial dilutions of PIs (100–1.56 pmol; right panels) immobilized on nitrocellulose membranes was analyzed by a protein-lipid overlay assay using an anti-GST antibody. Lysophosphatidic acid is denoted by LPA; lysophosphocholine is denoted by LPC; sphingosine-1-phosphate is denoted by SP; phosphatidic acid is denoted by PA; and phosphatidylserine is denoted by PS. The experiment was reproduced at least three times with similar results.</p><p>(B) PL vesicles (20 μl, 1 mM lipid) composed of PC (65%), PE (30%), and 5% (1 nmol) either PI(4)P, PI(3)P or PI(4,5)P₂ were incubated with affinity-purified GST-SidC or GST-SidD (40 pmol), centrifuged, and washed. Binding of GST fusion proteins to PL vesicles was assayed by Western blot with an anti-GST antibody. Similar results were obtained in three separate experiments.</p></div

PI3Ks Affect the Amount of SidC Bound to LCVs in <i>Dictyostelium</i>

<div><p>(A) Confocal laser scanning micrographs of calnexin-GFP–labeled <i>Dictyostelium</i> wild-type strain Ax3 (green), infected with DsRed-Express–labeled wild-type L. pneumophila (red) for 1 h (left panel), and immuno-labeled for SidC (blue) with an affinity-purified primary and Cy5-conjugated secondary antibody (middle panel). To quantify fluorescence intensity (right panel), the averaged fluorescence intensity of background areas (B1, B2, and B3) was subtracted from the intensity of the sample area (S). Bar denotes 2 μm.</p><p>(B) Dot plot of SidC fluorescence (average and variance) on LCVs within <i>Dictyostelium</i> wild-type (untreated, <i>n</i> = 135; 20 μM LY, <i>n</i> = 94) or Δ<i>PI3K1/2</i> (<i>n</i> = 86). The data shown are combined from six independent experiments, each normalized to 100% (average SidC fluorescence on LCVs in wild-type <i>Dictyostelium</i>).</p></div

Trafficking of L. pneumophila within <i>Dictyostelium</i> Lacking Functional PI3Ks

<div><p>(A) Confocal laser scanning micrographs of calnexin-GFP–labeled <i>Dictyostelium</i> (green) wild-type Ax3 (WT denotes untreated or treated with 20 μM LY) or Δ<i>PI3K1/2</i> (denoted by ΔPI3K) infected with DsRed-Express–labeled wild-type L. pneumophila (red) for 1 h or 2.5 h, respectively. Representative examples of spacious vacuoles (WT) and tight vacuoles (WT/LY, ΔPI3K) are shown. DNA was stained with DAPI (blue). Bar denotes 2 μm.</p><p>(B) Quantification over time of spacious LCVs in calnexin-GFP–labeled <i>Dictyostelium</i> wild-type (filled squares denote untreated; filled triangles denote LY) or Δ<i>PI3K1/2</i> (open squares) infected with DsRed-Express–labeled wild-type <i>L. pneumophila.</i></p><p>Data represent means and standard deviations of the percentage of spacious vacuoles from 50–200 total vacuoles per time point scored in four independent experiments.</p><p>(C) Confocal laser scanning micrographs of <i>Dictyostelium</i> wild-type or Δ<i>PI3K1/2</i>, infected for 75 min with wild-type L. pneumophila expressing M45-tagged SidC. Infected amoebae were stained with rhodamine-conjugated anti-L. pneumophila antibody (red), FITC-conjugated anti-M45-tag antibody (green), and DAPI (blue), respectively. Bar denotes 2 μm.</p></div

PI(4)P Is a Lipid Marker of LCVs Harboring Icm/Dot–Proficient L. pneumophila

<div><p>(A, B, and D) Confocal micrographs of LCVs in lysates of (A) calnexin-GFP–labeled <i>Dictyostelium,</i> (B) VatM-GFP–labeled <i>Dictyostelium,</i> or (D) RAW264.7 macrophages infected with DsRed-Express–labeled L. pneumophila are shown. The lysates were prepared with a ball homogenizer, and PI(4)P was visualized on the LCVs using as probes either the PH domain of the PI(4)P-binding protein FAPP1 fused to GST, an antibody against PI(4)P, or GST-SidC. Using GST alone or omission of the anti-PI(4)P antibody did not label the LCVs. Bar denotes 2 μm (magnification of all images is identical).</p><p>(C) Quantification of PI(4)P-positive calnexin-GFP–labeled (<i>n</i> = 300) or VatM-GFP–labeled (<i>n</i> = 100) LCVs in <i>Dictyostelium</i> wild-type strain Ax3.</p></div

Phagocytosis of Wild-Type L. pneumophila or Δ<i>icmT</i> by <i>Dictyostelium</i>

<p>Phagocytosis of <i>gfp</i>-expressing wild-type L. pneumophila (black bars) or Δ<i>icmT</i> (grey bars) by <i>Dictyostelium</i> infected at (A and B) an MOI of 100 or (C) at the MOI indicated was analyzed by flow cytometry. The increase in GFP fluorescence (FL1, <i>x</i>-axis) indicates that, at MOIs ranging from 1–100, the number of wild-type L. pneumophila phagocytosed is about one order of magnitude higher than the number of Δ<i>icmT.</i> Phagocytosis is blocked by latrunculin B or incubation at 4 °C. (B and C) The data shown are the means and standard deviations of duplicates and are representative of at least three independent experiments.</p

A Role for PI3Ks in Intracellular Replication but Not Phagocytosis of Wild-Type L. pneumophila

<div><p>(A) Phagocytosis by <i>Dictyostelium</i> wild-type Ax3 or Δ<i>PI3K1/2</i> (untreated or treated with 5 μM WM) of GFP-labeled L. pneumophila wild-type (black bars) or a Δ<i>icmT</i> mutant strain (grey bars) was determined by flow cytometry.</p><p>(B) Release of <i>L. pneumophila</i> wild-type (squares) or Δ<i>icmT</i> (circles) into the supernatant of <i>Dictyostelium</i> wild-type (denoted by filled symbols) or Δ<i>PI3K1/2</i> (denoted by open symbols) was quantified by CFUs.</p><p>(C) Release of wild-type <i>L. pneumophila</i> (CFUs) from <i>Dictyostelium</i> wild-type (filled squares denote untreated; filled triangles denote 10 μM LY) or Δ<i>PI3K1/2</i> (open squares denote untreated; open triangles denote LY).</p><p>(D) Quantification by flow cytometry of intracellular growth of GFP-labeled wild-type L. pneumophila within wild-type <i>Dictyostelium</i> or Δ<i>PI3K1/2</i> in the presence or absence of 20 μM LY.</p><p>The data shown are means and standard deviations of duplicates (A) or triplicates (B and C), and are representative of at least three independent experiments (A–D).</p></div

Effect of anti-virulence compounds on mycobacterial cell wall permeability.

<p>Mycobacteria were grown in the presence of each compound (10 μM), incubated for 20 min in the presence of 6μM ethidium bromide, and analyzed by flow cytometry. Analysis of fluorescence was performed on bacteria with very similar aggregate size. The average and SEM of five or six independent experiments is presented. One compound (M8) significantly inhibited bacterial permeability (one-way analysis of variance: p = 0.0002; *: post-hoc Tukey-Kramer p<0.05).</p

Effect of anti-virulence compounds on mycobacterial aggregation.

<p>In order to assess mycobacterial aggregation, cultures were analyzed by flow cytometry and the forward scatter (FSC) of bacterial aggregates was recorded. (A) Untreated cells (NT) aggregated readily, and as expected, repeated passage through a needle (NT + needle) reduced aggregation. Cultivation in the presence of compound M39 (10 μM) reduced the degree of aggregation. (B) In five independent experiments, the mean FSC of <i>M</i>. <i>marinum</i> cultures was measured, and expressed as a percentage of FSC measured in an untreated culture. Three compounds (M8, M24, M39) significantly inhibited mycobacterial aggregation (one-way analysis of variance: p = 0.0002; *: post-hoc Tukey-Kramer p<0.05).</p

Chemical libraries screened in this study.

<p>Chemical libraries screened in this study.</p