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

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

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 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