Fig. 2

The PltB- and PltC-typhoid toxins exhibit different biological properties. a Gel filtration chromatography and SDS-PAGE/Coomassie blue (inset) analyses of purified PltC-typhoid toxin. Lanes on gel represent individual chromatographic fractions (red box) containing purified toxin. b PltC-typhoid toxin elicits G2/M cell cycle arrest in human epithelial cells. Purified PltB- or PltC-typhoid toxins were added to the culture medium of Henle-407 cells at the indicated concentrations and 48h after, cells were fixed and analyzed by flow cytometry to evaluate toxicity as indicated in Materials and Methods. The data shown are the mean normalized toxicity ± S.D. for three independent experiments. c PltC-typhoid toxin does not induce G2/M arrest in S. Typhi-infected cells. Henle-407 cells were infected with the indicated strains at a multiplicity of infection (MOI) of 10 or 30, as indicated, and 48h post-infection cells were collected and the percentage of cells in G2/M phase was determined as described for panel b. Mean values±S.D. are shown for three independent experiments assayed in duplicate (6 total samples). Asterisks denote statistically significant levels G2/M cell cycle arrest compared to mock infected cells (red dotted line) as determined by unpaired two-tailed t-tests. d, e PltC-typhoid toxin is not packaged into vesicle transport carriers. Henle-407 cells were infected with the indicated S. Typhi strains encoding 3x-FLAG epitope-tagged CdtB and 48 hs post-infection the cells were fixed and stained with DAPI (blue), α-FLAG (green), and α-S. Typhi LPS (red) antibodies. Typhoid toxin-containing export vesicles, which appear as green puncta d, were quantified by image analysis e as indicated in Materials and methods. Values are from >25 images (~100 infected cells) taken in two independent experiments and represent the mean relative ratios ± S.E.M. Asterisks denote the statistical significance of the indicated pairwise comparisons determined using unpaired two-tailed t-tests. f–h The PltB- and PltC-typhoid toxins elicit different effects when administered to mice. Highly purified preparations of PltB- (2μg) or PltC-typhoid toxins (10μg) were administered to C57BL/6 mice. For one group of mice, their survival f and body weight (mean ± S.D.) g was recorded at the indicated times. The remaining mice were killed at four days post-toxin administration and a blood sample was collected and analyzed to quantify the indicated cell types (mean ± S.D.) h. WBCs, white blood cells. The Mantel-Cox test was used for statistical analysis of mouse survival and Brown-Forsythe and Welch ANOVA coupled with Dunnett’s T3 multiple comparisons tests were used to statistically compare the indicated samples for the blood analysis. For all panels, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s.s. not statistically significant. Source data are provided as a Source Data file