Inhibition of dynamin with dynasore and a dominant-negative mutant.

<p>Dynasore-pretreated and non-pretreated HeLa cells were incubated with (A) 5 nM diphtheria toxin for 180 min or (B) 4 pM toxin B for 75 min, prior to microscopical analysis of the cell morphology. (C) HeLa cells preincubated with dynasore or with solvent only were treated with 4 pM toxin B for 75 min, 1.5 nM toxin A for 105 min, 4 nM lethal toxin for 75 min or 1.5 nM α-toxin for 60 min. The percentage of rounded cells was quantified and data are given +/− SD (n = 3) and from a minimum of 200 cells in total. (D) Dynasore-pretreated HT-29 cells and untreated cells were intoxicated with 40 pM toxin. After onset of cell rounding (150 min), glucosylation status of Rac1 in cell lysates was analyzed with antibodies recognizing either only unmodified Rac1 (α-Rac1_non-Glc) or all Rac1 (α-Rac1_total). (E) HeLa cells expressing dominant-negative dynamin (HA-dynamin_K44A) for 24 h or mock-transfected cells were intoxicated with 4 pM toxin B for 75 min and cell morphology analyzed microscopically. (F) Selective expression of HA-dynamin_K44A in plasmid-transfected HeLa cells, but not in mock-transfected cells, was detected in cell lysates with an anti-HA antibody. Antibody detection of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) served as a control for equal loading of lysate samples.</p

RNAi-mediated gene silencing of the clathrin heavy chain.

<p>HeLa cells were transfected with siRNA against the clathrin heavy chain (si_clathrin). (A) Two days after transfection, cell lysates from si_clathrin-transfected cells and mock-transfected cells were analyzed for clathrin heavy chain expression using a specific antibody (α-clathrin(HC)). Antibody detection of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) served as a control for equal loading of lysate samples. (B) After addition of toxin B (4 pM), Rac1 glucosylation status was compared in lysates from si_clathrin- and mock-transfected HeLa cells at indicated time points with antibodies recognizing either only unmodified Rac1 (α-Rac1_non-Glc) or all Rac1 (α-Rac1_total).</p

Disintegration of lipid rafts by exogenous sphingomyelin depletion with SMase.

<p>HeLa cells were pretreated with SMase or left untreated prior to intoxication with (A) 4 pM toxin B for 75 min, 5 nM toxin A for 300 min, 5 nM lethal toxin for 180 min, 5 nM α-toxin for 120 min, (B) mock incubation for 300 min, or (C) 50 nM VacA toxin for 300 min. Images were obtained by microscopy, upon onset of intoxication characteristics. (D) SMase-preincubated or non-preincubated HeLa cells were intoxicated with 4 pM toxin B or left untreated. The percentage of cell rounding was quantified from three independent experiments at indicated time points (data are given +/− SD).</p

Pharmacological inhibition of clathrin assembly with chlorpromazine.

<p>HeLa cells were preincubated with chlorpromazine (Cp) or left untreated, prior to addition of (A) 5 nM diphtheria toxin (DT) and incubation for 180 min, (B) 3.5 nM CNF1 and incubation for 150 min or (C) 4 pM toxin B and incubation for 75 min. (D) The percentage of cell rounding in chlorpromazine-pretreated or non-pretreated HeLa cells after intoxication with 4 pM toxin B for 75 min, 1.5 nM toxin A for 105 min, 4 nM lethal toxin for 75 min or 1.5 nM α-toxin for 60 min was quantified and data are given +/− SD (n = 3) and from a minimum of 200 cells in total. (E) Human intestinal epithelial cells (CaCo-2) were grown to confluency on filters and were preincubated with chlorpromazine (Cp) or left untreated. A subset of cells was intoxicated with 40 pM toxin B and transepithelial electrical resistance (TER) was measured at indicated time points, where starting resistance was set to 100% and TER values are calculated as relative TER in % from starting resistance (+/− SD, n = 3).</p

Expression of dominant-negative Eps15 and Cav-1.

<p>(A) HeLa cells, transfected with plasmids, encoding dominant-negative forms of Eps15 or Cav-1 fused to a GFP moiety, were eventually intoxicated for 75 min with 4 pM toxin B or left untreated. Toxin B-induced alterations in cell morphology were analyzed in Eps15- (Eps15 DN::EGFP), Cav-1- (GFP::Cav-1 DN) and mock-transfected cells after actin staining with FITC-phalloidin (red and grey signals) and by applying fluorescence microscopy. Green signals derived from cells expressing GFP-tagged, dominant-negative Eps15 or Cav-1, respectively. (B) HeLa cells were transfected with a plasmid encoding Eps15 DN::EGFP, intoxicated with 4 pM toxin B for 30 min and subsequently subjected to fluorescence-assisted cell sorting. GFP excitation of the Eps15 DN::EGFP-transfected cells resulted in two cell populations (left panel, blue curve, bordered with dashed line). One population represents non-transfected cells, with overlapping background fluorescence as obtained in mock-transfected cells (left panel, red curve). The other population represents Eps15 DN::EGFP-expressing cells. Equal number of cells from both cell populations (non-transf. and Eps15 DN::EGFP) where subjected to cell lysis and analysis of the Rac1 glucosylation status with antibodies recognizing either only unmodified Rac1 (α-Rac1_non-Glc) or all Rac1 (α-Rac1_total). (C) Procedure was performed essentially as described in (B), but with HeLa cells transfected with a plasmid encoding GFP::Cav-1.</p

CNF1 binds to the cell surface via a C-terminal peptide.

<p>Suspensions of HeLa cells (1×10⁵ cells in 1 ml medium) were incubated for 20 min at 4°C with 2 µg DyLight488-labeled GST-CNF1 (CNF1DL488) alone, or together with increasing amounts (up to 30 fold) of untagged non-labeled CNF1 and the non-labeled fragments GST-CNF1-(426–1014), GST-CNF1-(720–1014), GST-CNF1-(709–1014) and GST-CNF1-(1–343), respectively. Following washing with PBS, cells were subjected to FACS analysis. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003884#s2" target="_blank">Results</a> are presented as mean of cell surface-bound fluorescence (% of control) of three independent experiments+standard deviation.</p

<i>Pasteurella Multocida</i> Toxin Prevents Osteoblast Differentiation by Transactivation of the MAP-Kinase Cascade via the Gα_q/11 - p63RhoGEF - RhoA Axis

<div><p>The 146-kDa <i>Pasteurella multocida</i> toxin (PMT) is the main virulence factor to induce <i>P. multocida</i>-associated progressive atrophic rhinitis in various animals. PMT leads to a destruction of nasal turbinate bones implicating an effect of the toxin on osteoblasts and/or osteoclasts. The toxin induces constitutive activation of Gα proteins of the G_q/11-, G_12/13- and G_i-family by deamidating an essential glutamine residue. To study the PMT effect on bone cells, we used primary osteoblasts derived from rat calvariae and stromal ST-2 cells as differentiation model. As marker of functional osteoblasts the expression and activity of alkaline phosphatase, formation of mineralization nodules or expression of specific transcription factors as osterix was determined. Here, we show that the toxin inhibits differentiation and/or function of osteoblasts by activation of Gα_q/11. Subsequently, Gα_q/11 activates RhoA via p63RhoGEF, which specifically interacts with Gα_q/11 but not with other G proteins like Gα_12/13 and Gα_i. Activated RhoA transactivates the mitogen-activated protein (MAP) kinase cascade via Rho kinase, involving Ras, MEK and ERK, resulting in inhibition of osteoblast differentiation. PMT-induced inhibition of differentiation was selective for the osteoblast lineage as adipocyte-like differentiation of ST-2 cells was not hampered. The present work provides novel insights, how the bacterial toxin PMT can control osteoblastic development by activating heterotrimeric G proteins of the Gα_q/11-family and is a molecular pathogenetic basis for understanding the role of the toxin in bone loss during progressive atrophic rhinitis induced by <i>Pasteurella multocida</i>.</p></div

Direct rBCAM-CNF interaction.

<p>A) For dot-blots 5 µl of 3 µM solutions of GST-CNFs, GST-CNF fragments and GST alone, respectively, were spotted onto a nitrocellulose membrane. The membrane was blocked with skimmed milk and recombinant BCAM (6 µM) was added for 1 h at room temperature. Following washing bound rBCAM was detected with an anti-Lu/BCAM antibody. Equal protein load was analyzed by visualizing the GST part of the spotted proteins with an anti GST-antibody. B) Biacore protein-protein interaction studies: An antibody against human IgG (Millipore) was coupled to two lanes of a CM5-biacore chip. As ligand recombinant BCAM containing a C-terminal human IgG domain (Sino biologics) was exclusively guided over lane 2. In a second step, GST-CNF proteins as analyte were guided over both lanes. Bound protein is given as relative units (RU) corrected for the unspecific binding to lane 1 as average plus standard deviation of three independent experiments.</p

Susceptibility of <i>S</i>. <i>cerevisiae</i> containing different actin variants towards iota toxin component Ia.

<p>Yeast strains containing wild type actin or actin variants with substitutions R177K, D179E, D179A, and D179K (panel A) or E270D and E270Q (panel B) were transformed with the vector alone or the Ia-expressing plasmid, and were analyzed by the drop-test under <i>ia</i>-repressing (glucose) or -inducing (galactose) conditions. Plates were incubated for 3–4 days at 30°C.</p

Competition and colocalization studies with HeLa cells.

<p>GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to HeLa cells. Following 2 h incubation the cells were lysed and the CNF1-catalysed deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting (A). HeLa cells were incubated with an anti Lu/BCAM antibody (AB B12) that binds to the extracellular domain or as control with an anti-Lu/BCAM antibody (AB C16) directed against the intracellular part of the glycoprotein. GST-CNF1 was then added to the cells for 2 h. We followed the toxins uptake by the amount of modified RhoA (shift in SDS-PAGE, B). Shown is a typical result of 3 independent experiments. Colocalization of DyLight488-labeled GST-CNF1 with Lu/BCAM and EEA1 (C) Top: HeLa-cells were treated on ice with DyLight488-labeled GST-CNF1 (5 mg/ml) (green) for 30 min to allow receptor binding. After 30 min cells were transferred to 37° for 30 min to induce uptake. Subsequently cells were fixed and stained for EEA1 (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a maximal projection of all confocal planes. Scale bar indicates 10 µm. Middle: HeLa-cells were treated with labeled GST-CNF1 as in A. After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Bottom: HeLa-cells were treated as in A, but instead of GST-CNF1, cells were treated with DyLight488-labeled GST-CNFY (5 mg/ml) (green). After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Shown is a typical staining of 9 HeLa cells analyzed.</p