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

Substrate Binding Analysis of the 23S rRNA Methyltransferase RrmJ

The 23S rRNA methyltransferase RrmJ (FtsJ) is responsible for the 2′-O methylation of the universally conserved U2552 in the A loop of 23S rRNA. This 23S rRNA modification appears to be critical for ribosome stability, because the absence of functional RrmJ causes the cellular accumulation of the individual ribosomal subunits at the expense of the functional 70S ribosomes. To gain insight into the mechanism of substrate recognition for RrmJ, we performed extensive site-directed mutagenesis of the residues conserved in RrmJ and characterized the mutant proteins both in vivo and in vitro. We identified a positively charged, highly conserved ridge in RrmJ that appears to play a significant role in 23S rRNA binding and methylation. We provide a structural model of how the A loop of the 23S rRNA binds to RrmJ. Based on these modeling studies and the structure of the 50S ribosome, we propose a two-step model where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2′-O position of the ribose

The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signaling and gene expression of Legionella pneumophila

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signaling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by [γ-(32) P]-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAs rsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signaling

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 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 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

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