Construction of pInv-H1.

<p>A. Organization of the inverted H1 promoters and sequence encoding a specific siRNA. The expression cassette contains essential promoter elements. The <i>Eco</i>RI and <i>Bgl</i>II sites flank the siRNA sequence and can be used to exchange siRNA cassettes. B. Generation of the Inv-H1-siRNA cassette by a ligase chain reaction (LCR). The numbers associated with the LCR-1 and LCR-2 products correspond to individual oligonucleotides that are ligated together. Digesting LCR-1 and LCR-2 with <i>Eco</i>RI allows for ligation and formation of the full-length product that can be cut with <i>Pst</i>I and <i>Hind</i>III and cloned into pSUPER. C. Gel electrophoresis of LCR-1 and LCR-2 products. Ten microliters of the LCR reaction were run in each lane of a 3% Metaphor agarose gel. The fragments corresponding to 106 bp (LCR-1) and 151 bp (LCR-2) were gel purified. D. Cloning scheme used to generate pInv-H1. The pSUPER construct and the LCR-derived product were digested with <i>Pst</i>I and <i>Hind</i>III and ligated together. The original full-length H1 promoter driving expression of the shRNA product in pSUPER has now been replaced with the inverted H1 promoter siRNA cassette.</p

Structure–Activity Relationship of Semicarbazone EGA Furnishes Photoaffinity Inhibitors of Anthrax Toxin Cellular Entry

EGA, <b>1</b>, prevents the entry of multiple viruses and bacterial toxins into mammalian cells by inhibiting vesicular trafficking. The cellular target of <b>1</b> is unknown, and a structure–activity relationship study was conducted in order to develop a strategy for target identification. A compound with midnanomolar potency was identified (<b>2</b>), and three photoaffinity labels were synthesized (<b>3</b>–<b>5</b>). For this series, the expected photochemistry of the phenyl azide moiety is a more important factor than the IC₅₀ of the photoprobe in obtaining a successful photolabeling event. While <b>3</b> was the most effective reversible inhibitor of the series, it provided no protection to cells against anthrax lethal toxin (LT) following UV irradiation. Conversely, <b>5</b>, which possessed weak bioactivity in the standard assay, conferred robust irreversible protection vs LT to cells upon UV photolysis

Intoxication Mediated by CdtA and CdtC Subunits.

<p>Jurkat, HeLa, or CHO-A745 cells were seeded in clear-bottom 384-well plates, incubated overnight, then challenged with the indicated toxin concentrations. Holotoxin, black circles; CdtAB, red squares; CdtBC, blue triangles. Intoxication was allowed to proceed for 48 h (Jurkat) or 72 h (HeLa and CHO-A745). Cell viability was measured by ATPlite reagent (Perkin Elmer), and normalized to ATPlite signal from unintoxicated controls. Data represent average values from three independent experiments, each performed in triplicate, +/- standard deviation. Lines represent nonlinear curve fit calculated using Prism 5 (GraphPad).</p

Tissue Culture LD₅₀ Values for Ec-Cdt Dimers and Trimers.

<p>Average values and standard deviation (+/-) were determined from at least three biological replicates, each performed in triplicate. NT, not tested; ND, value not determined due to lack of cytotoxicity.</p><p>Tissue Culture LD₅₀ Values for Ec-Cdt Dimers and Trimers.</p

Tissue Culture LD₅₀ Values for Hd-Cdt Dimers and Trimers.

<p>Average values and standard deviation (+/-) were determined from at least three biological replicates, each performed in triplicate. NT, not tested; ND, value not determined due to lack of cytotoxicity.</p><p>Tissue Culture LD₅₀ Values for Hd-Cdt Dimers and Trimers.</p

Holotoxin Assembly Method Affects Sensitivity to EGA.

<p>CHO-A745 cells were intoxicated as above in the presence or absence of 12.5 μM EGA. Additionally, cells were challenged with a combination of purified CdtA, CdtB, and CdtC subunits that were combined at the time of intoxication without further purification of assembled holotoxin (Ec-ABC). Cell viability was measured by ATPlite and normalized as above. Data represent average values from three independent experiments, each performed in triplicate.</p

CdtC Mediates Cholesterol Dependency of Ec-CDT.

<p>CHO-A745 cells were seeded at 8 x 10³ cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with or without 5 mM MβCD and/or 12.5 μM EGA for 1 h then challenged with 1 μM Ec-CDT or Ec-CdtAB for 16 h. Intoxication was assessed by measuring pH₂AX by laser scanning cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143977#pone.0143977.g002" target="_blank">Fig 2B</a>. Data were normalized against pH₂AX signal induced by Ec-CDT holotoxin (maximum signal) in each experiment. Graphs represent average values and SEM from three independent experiments, each performed in triplicate. All statistical analyses are from the pairwise post-test (Tukey’s) derived from one-way ANOVA. (Prism 5, GraphPad). Symbols above each column reflect comparison to Ec-CDT holotoxin (ns = not significant; * p < 0.001). Additional pairwise comparisons are indicated by brackets.</p

Ec-CdtC Dictates Resistance to EGA and Alters Intracellular Trafficking of Ec-CdtB.

<p>(A) CHO-A745 cells were intoxicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143977#pone.0143977.g001" target="_blank">Fig 1</a> except that all wells were additionally treated with 12.5 μM EGA. (B) CHO-A745 cells were seeded at 8 x 10³ cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with 1μM Ec-CDT holotoxin or 1 μM Ec-CdtAB for 4 or 16 h. Phosphorylated H₂AX (anti-pH₂AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH₂AX induced by Ec-CDT holotoxin was set at 100% and used to normalize signal from CdtAB for each time point. Graphs represent average values from three independent experiments, each performed at least 3 times. *p value = 0.0121 calculated by unpaired two-tailed t test (Prism 5, GraphPad). (C, D) CHO-A745 cells were seeded at 2 x 10⁴ cells/well on 8-well chambered slides and allowed to adhere overnight. The next day, cells were incubated on ice with 100 μM Ec-CDT holotoxin, Ec-CdtAB or Ec-CdtBC for 30 min, washed and incubated at 37°C for 60 minutes. Cells were then fixed, stained, and imaged as described in Methods [anti-Ec-CdtB (green) and EEA1 or Rab9 antibody (red)]. White scale bars at the left panel of each treatment indicate 10 μm and the right insert panel indicate 2 μm. Quantification of microscopy results was performed using Pearson's coefficient values indicating colocalization of the Ec-CdtB signal with the EEA1 or Rab9 enriched vesicles. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.</p

Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry

<div><p>Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by <i>Haemophilus ducreyi</i> (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.</p></div

The interaction of Derl2 and p97 is not required for CDT intoxication.

<p>(a) Derl2-GFP fails to bind p97, similar to Derl2ΔC. 293 cells were transfected with vectors encoding S-tagged versions of the indicated forms of Derl2. After 3 days, the cells were lysed and western blot was performed on S-protein precipitates with anti-p97 and anti-S-tag antibodies (b) Overexpression of Derl2-GFP does not affect Hd-CDT intoxication of parental A745TKR cells. Parental A745TKR cells expressing empty vector, Derl2 or Derl2-GFP were intoxicated with Hd-CDT, similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004295#ppat-1004295-g001" target="_blank">Fig. 1</a>. (c, d) Derl2-GFP and Derl2ΔC complement sensitivity to Hd-CDT in CHO-CDT^RC1. CHO-CDT^RC1 cells expressing empty vector, Derl2, (c) Derl2-GFP or (d) Derl2ΔC were intoxicated similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004295#ppat-1004295-g001" target="_blank">Fig. 1</a>. (e) Dominant negative p97 reduces sensitivity of 293 cells to Hd-CDT. 293 cells stably expressing TCRαGFP were transfected with plasmids encoding CD4 and either dominant negative (R586A) or control (R700A) p97, followed by intoxication with Hd-CDT for 48 hours and staining with Hoechst and anti-CD4 antibodies. Flow cytometry was performed to obtain geometric mean fluorescence values for TCRαGFP (GFP) in CD4+ cells and cell cycle profile of CD4 negative (grey shaded; untransfected control) and CD4 positive cells (black lines). (f) The Derl2 WR motif is not required for intoxication by Hd-CDT. CHO-CDT^RC1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A or Derl2 T59A were intoxicated similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004295#ppat-1004295-g001" target="_blank">figure 1</a>. (g–i) Retrograde trafficking of Hd-CDT in p97 deficient cells is blocked at the endoplasmic reticulum. (g) Following transfection with pH2B-GFP (blue) and either dominant negative or control p97, wildtype and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with anti-Hd-CdtB (green) antibody and anti-calreticulin antibody (red). White scale bars indicate 5 µm. pH2B-GFP pseudo-colored blue; Hd-CdtB pseudo-colored green and calreticulin pseudo-colored red (h, i) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments, percent viability is normalized to unintoxicated controls and error bars indicate standard error.</p