Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9

Characterization of cellulolytic activity in the gut of the terrestrial land slug Arion ater : Biochemical identification of targets for intensive study

The level of cellulolytic activity in different areas of the gut of the terrestrial slug Arion ater was assayed at different temperatures and pH values. To do this, crude gut proteins were isolated and assayed using modified dinitrosalicylic acid reducing sugar assay. Crude protein samples were also separated and cellulolytic activity identified using in gel CMC zymography and esculin hydrate activity gel assays. pH and temperature profiling revealed optimum cellulolytic activity between pH5.0 and 6.0 for different gut regions and retention of up to 90% of activity at temperatures up to 50°C. Zymograms and activity gels revealed multiple endoglucanase and β-glucosidase enzymes. To further investigate the source of this cellulolytic activity bacterial isolates from the gut were tested for endoglucanase and β-glucosidase activity using growth plate assays. 12 cellulolytic microbes were identified using 16S rDNA gene sequencing. These include members of the genera Buttiauxella, Enterobacter, Citrobacter, Serratia and Klebsiella. Gut metagenomic DNA was then subjected to PCR, targeting a 400bp region of the 16SrDNA gene which was subsequently separated and individuals identified using DGGE. This identified members of the genera Citrobacter, Serratia, Pectobacterium, Acinetobacter, Mycoplasma, Pantoea and Erwinia. In summary, multiple glycoside hydrolase enzymes active over a broad range of temperature and pH values in a relatively under studied organism were detected, indicating that the gut of A. ater is a viable target for intensive study to identify novel carbohydrate active enzymes that may be used in the biofuel industry

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

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 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

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

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

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