11 research outputs found
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
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
Tissue Culture LD<sub>50</sub> 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 LD<sub>50</sub> Values for Ec-Cdt Dimers and Trimers.</p
Tissue Culture LD<sub>50</sub> 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 LD<sub>50</sub> Values for Hd-Cdt Dimers and Trimers.</p
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<sup>R</sup>C1. CHO-CDT<sup>R</sup>C1 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<sup>R</sup>C1 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
Derl2 is required for CDT intoxication.
<p>Viability of parental A745TKR cells, retrovirally induced mutant CHO-CDT<sup>R</sup>C1 cells, and CHO-CDT<sup>R</sup>C1 cells expressing Derl2 after intoxication with Aa-CDT (a), Hd-CDT (b), Ec-CDT (c) and Cj-CDT (d). Intoxication was performed 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) Top: representation of the <i>Derl2</i> open reading frame with boxes representing exons, gray arrows representing primers, and upside down triangles representing proviral insertions. Bottom: agarose gel of genomic PCR from parental A745TKR, CHO-CDT<sup>R</sup>C1 and CHO-CDT<sup>R</sup>F1 cells using primers detailed in the diagram. (f) Overexpression of Derl1 does not complement resistance to CDT. Derl2 deficient CHO-CDT<sup>R</sup>C1 cells expressing empty vector, Derl1, and Derl2 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>. (g) Derl2 was immunoprecipitated from normalized cell lysates and precipitated proteins analyzed by western blot with anti-Derl2 antibody. (h) CRISPR mediated deletion of Derl2 in HeLa cells causes resistance to Hd-CDT. HeLa cells were transfected with Cas9 DNA and gDNA, followed by selection with G418 and Hd-CDT. Following selection, wildtype and Derl2-deleted cells 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">figure 1</a>. (i) CRISPR mediated deletion of Derl2 results in decreased expression as judged by western blot of anti-Derl2 immunoprecipitated protein from normalized cell lysates. Increasing amounts of immunoprecipitated protein loaded for each condition, corresponding to input from 0.5, 1, or 2×10<sup>6</sup> cells. (j–l) Retrograde trafficking of Hd-CDT in Derl2 deficient cells is blocked at the endoplasmic reticulum. (j) A745TKR and CHO-CDT<sup>R</sup>C1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 10 or 60 minutes. Cells were then fixed and stained with DAPI (nuclei, blue), Concanavalin A (ER, red) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (k,l) 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 marker. 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
CdtC Mediates Cholesterol Dependency of Ec-CDT.
<p>CHO-A745 cells were seeded at 8 x 10<sup>3</sup> 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 pH<sub>2</sub>AX 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 pH<sub>2</sub>AX 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 10<sup>3</sup> 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 H<sub>2</sub>AX (anti-pH<sub>2</sub>AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH<sub>2</sub>AX 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 10<sup>4</sup> 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
Derl2 and Hrd1 contribute to sensitivity to Ricin, independent of the Derl2 WR motif and the interaction of Derl2 with p97.
<p>(a) Derl2 deficiency causes resistance to ricin. A745TKR cells, CHO-CDT<sup>R</sup>C1 cells, and CHO-CDT<sup>R</sup>C1 cells expressing Derl2 were seeded in a 384-well plate (1×10<sup>3</sup> cells/well) and allowed to adhere overnight, followed by 48 hour intoxication with ricin and quantitation of viability using ATPlite 1-step reagent (Perkin Elmer). Ricin LD<sub>50</sub> values were calculated from three independent experiments and paired t-test was performed to calculate two tailed p-values. (b) CRISPR mediated Hrd1 deletion in 293 cells causes resistance to ricin. Wildtype and Hrd1-deleted 293 cells were intoxicated with ricin, similar to figure (a). (c) Derl2ΔC complements the resistance to ricin. CHO-CDT<sup>R</sup>C1 cells expressing empty vector, Derl2 and Derl2ΔC were intoxicated similar to (a). (d) The Derl2 WR motif is not required for intoxication by ricin. CHO-CDT<sup>R</sup>C1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A and Derl2 T59A were intoxicated similar to (a). Data are representative of at least three independent experiments performed in triplicate, percent viability is normalized to unintoxicated controls and error bars indicate standard error.</p
Identification of Derl2 domains required for CDT intoxication.
<p>(a–c) CHO-CDT<sup>R</sup>C1 cells expressing empty vector (squares), Derl1-S (triangles), or Derl2-S (diamonds) were intoxicated in each panel, similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004295#ppat-1004295-g001" target="_blank">Fig. 1</a>, and compared to derlin variants indicated below. Anti-DERL1 (a) or anti-Derl2 (b, c) western blot of S-protein agarose precipitated protein from normalized cell lysates show expression levels of chimeric derlins. Cartoons depict Derl1 (black) and Derl2 (grey) sequences in each chimera. (a) CHO-CDT<sup>R</sup>C1 cells expressing Derl1-S (triangles, #1) or Derl2<sup>1–187</sup>:Derl1<sup>189–251</sup>-S tag (circles, #2) were challenged with Hd-CDT. (b) CHO-CDT<sup>R</sup>C1 cells expressing Derl2-S (diamonds, #1), Derl2<sup>1–112</sup>:Derl1<sup>114–121</sup>:Derl2<sup>120–239</sup>-S (circles, #2) or Derl2<sup>1–161</sup>:Derl1<sup>163–171</sup>: Derl2<sup>171–239</sup>-S (inverted triangles, #3) were intoxicated as above. (c) CHO-CDT<sup>R</sup>C1 cells expressing Derl2-S (diamonds, #1), Derl1<sup>1–88</sup>:Derl2<sup>88–239</sup>-S (open boxes, 2), Derl1<sup>1–138</sup>:Derl2<sup>138–239</sup>-S (open triangles, #3) or Derl1<sup>1–162</sup>:Derl2<sup>162–239</sup>-S (open diamonds, #4) were intoxicated as above. Data are representative of at least three independent experiments performed in triplicate, percent viability is normalized to unintoxicated controls and error bars indicate standard error.</p