Reaction Mechanism of Adenylyltransferase DrrA from <i>Legionella pneumophila</i> Elucidated by Time-Resolved Fourier Transform Infrared Spectroscopy

Modulation of the function of small GTPases that regulate vesicular trafficking is a strategy employed by several human pathogens. <i>Legionella pneumophila</i> infects lung macrophages and injects a plethora of different proteins into its host cell. Among these is DrrA/SidM, which catalyzes stable adenylylation of Rab1b, a regulator of endoplasmatic reticulum to Golgi trafficking, and thereby alters the function and interactions of this small GTPase. We employed time-resolved FTIR-spectroscopy to monitor the DrrA-catalyzed AMP-transfer to Tyr77 of Rab1b. A transient complex between DrrA, adenylylated Rab1b, and the pyrophosphate byproduct was resolved, allowing us to analyze the interactions at the active site. Combination of isotopic labeling and site-directed mutagenesis allowed us to derive the catalytic mechanism of DrrA from the FTIR difference spectra. DrrA shares crucial residues in the ATP-binding pocket with similar AMP-transferring enzymes such as glutamine synthetase adenylyltransferase or kanamycin nucleotidyltransferase, but provides the complete active site on a single subunit. We determined that Asp112 of DrrA functions as the catalytic base for deprotonation of Tyr77 of Rab1b to enable nucleophilic attack on the ATP. The study provides detailed understanding of the <i>Legionella pneumophila</i> protein DrrA and of AMP-transfer reactions in general

Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity

Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity

Activation of Ran GTPase by a <i>Legionella</i> Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

<div><p>The causative agent of Legionnaires' disease, <i>Legionella pneumophila</i>, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “<i>Legionella</i>-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of <i>L. pneumophila</i>. Moreover, the <i>L. pneumophila</i> protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. <i>L. pneumophila</i> wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. <i>L. pneumophila</i> lacking <i>legG1</i> is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a <i>Yersinia</i> strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type <i>L. pneumophila</i> rapidly move along microtubules, while LCVs harboring Δ<i>legG1</i> mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of <i>L. pneumophila</i>.</p></div

LegG1 stabilizes microtubules in <i>L. pneumophila</i>-infected phagocytes.

<p>(<b>A, B</b>) Microtubules were analyzed by confocal laser scanning fluorescence microscopy in (<b>A</b>) <i>D. discoideum</i> producing tubulin-GFP or (<b>B</b>) RAW264.7 macrophages infected (MOI 10, 2 h (amoebae), 4 h (macrophages)) with DsRed-producing <i>L. pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>legG1</i> harboring pCR077 or with Δ<i>legG1</i>/pER5 (M45-LegG1). The macrophages were immuno-labeled for α-tubulin (green) and SidC (blue) and, as controls, treated with taxol or nocodazole (30 µM). (<b>C, D</b>) Microtubules were analyzed by STED microscopy in RAW264.7 macrophages infected (MOI 10, 4 h) with GFP-producing <i>L. pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>legG1</i> harboring pCR076 or with Δ<i>legG1</i>/pER4 (M45-LegG1) and immuno-labeled for (<b>C</b>) α-tubulin (grey), or (<b>D</b>) α-tubulin (green) and SidC (blue). Uninfected macrophages were treated with taxol or nocodazole (30 µM) as control. Bars, 1 µm (A), 5 µm (B, C), 0.5 µm (D). (<b>E</b>) Microtubule polymerization was analyzed by anti-tubulin Western blot in RAW264.7 macrophages infected (MOI 10, 4 h) with GFP-producing <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). As a control, uninfected macrophages were treated with taxol or nocodazole (30 µM). Homogenates of the macrophages were centrifuged (20'000×<i>g</i>, 30 min) to separate polymerized tubulin in the pellet (P) from soluble tubulin in the supernatant (S), and the amount of microtubule polymerization was assessed by α-tubulin Western blot (upper panel). Actin was used as a control. A representative experiment is shown, and the ratio of polymerized to soluble α-tubulin is indicated (R). The graph (lower panel) shows means and standard deviations of 3 independent experiments.</p

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.</p

Microbial microinjection of LegG1 promotes microtubule polymerization.

<p>(<b>A</b>) <i>Yersinia enterocolitica</i> strain WA (pT3SS), encoding the Ysc T3SS but lacking type III-secreted effectors, produced and secreted fusion proteins of YopE_1–53 or YopE_1–138 with the <i>Legionella</i> Ran activator LegG1 or the Rab1 GEF SidM. The proteins in the pellet (P) or supernatant (S) were precipitated by chloroform/methanol treatment and visualized by Western blot using an antibody against YopE. (<b>B</b>) HeLa cells were infected (MOI 10, 2 h) with <i>Y. enterocolitica</i> WA (pT3SS) producing YopE_1–53-LegG1, washed several times and lysed with 1% digitonin. After centrifugation, proteins in the pellet (intact bacteria, debris) and in the supernatant (translocated bacterial/soluble host proteins) were precipitated, and the fusion protein was visualized by Western blot using an anti-YopE antibody. Controls: HeLa cells alone, bacteria treated with 1% digitonin, 1% SDS or with the T3SS inhibitor CCCP (50 µM). (<b>C</b>) Fluorescence microscopy of YopE_1–138-LegG1 translocation into HeLa cells infected (MOI 10, 2 h) with <i>Y. enterocolitica</i> WA (pT3SS) producing YopE_1–138-LegG1, YopE_1–138-SidM or YopE. Controls: uninfected cells or cells treated with 30 µM taxol or nocodazole. The cells were immuno-stained for α-tubulin and YopE, and nuclei were labeled with DAPI. Bars, 10 µm. (<b>D</b>) Fluorescence microscopy of YopE_1–53-LegG1 translocation into HeLa cells treated with nocodazole (1 µM, 1 h) and infected (MOI 10, 2 h) with <i>Y. enterocolitica</i> WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1. The cells were immuno-stained for α-tubulin (green) and YopE (red), nuclei were labeled with DAPI (grey). Bars, 40 µm or 10 µm (insets). (<b>E</b>) Western blot of YopE_1–53-LegG1 translocation into HeLa cells treated with nocodazole (1 µM, 1 h) and infected (MOI 10, 2 h) with <i>Y. enterocolitica</i> WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1. The soluble microtubule fraction in the cell supernatant (S) and the pellet (P) was analyzed with an anti-α-tubulin antibody; ratio of polymerized to soluble α-tubulin (R). (<b>F</b>) Fluorescence microscopy of A549 cells treated or not with siRNA against Ran and infected (MOI 10, 2 h) with <i>Y. enterocolitica</i> WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1. The cells were immuno-stained for α-tubulin (green) and YopE (red), and nuclei were labeled with DAPI (grey). Bars, 10 µm or 5 µm (insets). The data shown is representative of 3 independent experiments (A–F).</p

LCVs harboring <i>L. pneumophila</i> Δ<i>legG1</i> show impaired motility.

<p>(<b>A</b>) Real-time fluorescence microscopy of LCV motility in <i>D. discoideum</i> producing calnexin-GFP and infected (MOI 10, 2 h) with DsRed-producing <i>L. pneumophila</i> wild-type or Δ<i>legG1</i> mutant bacteria harboring pSW001. Two hours post infection, trafficking of LCVs was recorded by laser confocal scanning microscopy for 5 min with images taken every 15 s. Bars, 1 µm. (<b>B</b>) The velocity of LCVs was quantified by tracking the migration distance of LCVs over time (n = 50/strain; ***, <i>p</i><0.001).</p

<i>L. pneumophila</i> wild-type but not Δ<i>legG1</i> activates Ran on LCVs.

<p>(<b>A</b>) LegG1 promotes RanBP1 accumulation on LCVs. <i>D. discoideum</i> producing RanBP1-GFP (green) was infected (MOI 50, 1 h) with DsRed-producing <i>L. pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>legG1</i> harboring pCR077 (red), or with Δ<i>legG1</i>/pER5 (M45-LegG1) and immuno-stained for SidC (blue). The percentage of RanBP1-GFP-positive LCVs (n = 100/strain, 5 independent experiments) was scored in lysates of infected cells (**, <i>p</i><0.01; ***, <i>p</i><0.001). (<b>B</b>) Production of Ran(GTP) in infected macrophages. RAW264.7 macrophages were infected (MOI 25, 1 h) with <i>L. pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>legG1</i> harboring pCR033 (vector) or with Δ<i>legG1</i>/pSU19 (M45-LegG1). The infected macrophages were lysed, activated Ran was immuno-precipitated with an antibody specifically recognizing Ran(GTP) and visualized by Western blot using an anti-Ran antibody. Lysates of uninfected cells incubated with GTP or GDP in presence of EDTA were used as positive or negative controls for endogenous GEF activity. Loading control: Western blot of Ran in samples before immuno-precipitation. (<b>C</b>) Production of Ran(GTP) in cell lysates. A549 epithelial cells were lysed and incubated with purified His₆-LegG1 (native or heat-inactivated, h. i.) in presence of excess GTP (100 µM) or GDP (1 mM). Activated Ran was immuno-precipitated with an antibody specifically recognizing Ran(GTP) and visualized by Western blot using an anti-Ran antibody. Loading control: Western blot of Ran in samples before immuno-precipitation. The relative amount of Ran(GTP) was determined by densitometry; means and standard deviations of 4 (B, C) independent experiments are shown (*, <i>p</i><0.05; **, <i>p</i><0.01).</p

The small GTPase Ran and the Icm/Dot substrate LegG1 localize to LCVs.

<p>(<b>A</b>) Ran accumulates on LCVs. <i>D. discoideum</i> producing RanA-GFP was infected (MOI 50, 1 h) with DsRed-producing <i>L. pneumophila</i> wild-type, Δ<i>legG1</i> or Δ<i>icmT</i> harboring pSW001 and immuno-stained for the LCV membrane marker SidC. LCVs in lysates of infected cells are shown. (<b>B</b>) Depletion of Ran or RanBP1 inhibits intracellular growth of <i>L. pneumophila</i>. A549 lung epithelial carcinoma cells were treated with AllStars siRNA (negative control) or with siRNA oligonucleotides targeting Ran, RanBP1 or Arf1 (positive control) for 2 d, and intracellular replication of GFP-producing <i>L. pneumophila</i> harboring pNT28 was quantified by fluorescence measurements after 24 h. Data represent mean and standard deviation of three independent experiments considering the 3 most effective out of 4 different oligonucleotides (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003598#ppat.1003598.s001" target="_blank">Fig. S1B</a>). Student's t-test; ***, <i>p</i><0.001. (<b>C</b>) Icm/Dot-dependent localization of M45-LegG1 on LCVs in cell homogenates. <i>D. discoideum</i> producing calnexin-GFP was infected (MOI 50) with <i>L. pneumophila</i> wild-type, Δ<i>icmT</i> or Δ<i>legG1</i> harboring pSU19 (M45-LegG1) or with Δ<i>legG1</i> harboring pCR033 (vector), homogenized and immuno-stained with an anti-M45 antibody and with DAPI. The percentage of M45-LegG1-positive LCVs (n = 100/strain, 3 independent experiments) was scored in lysates of infected cells (*, <i>p</i><0.05). Bars (A, C), 0.5 µm.</p