Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen
Fig 5
Characterization of typhoid toxin trafficking to the endoplasmic reticulum and cytosol in CRISPR/Cas9-edited cell lines.
(A) Wild-type and knockout cells lines were treated with purified typhoid toxin and at the indicated time points, typhoid toxin was recovered from cell lysates by affinity chromatography and analyzed by western blot with an anti toxin antibody as indicated in Materials and Methods. (B) Proportion of typhoid toxin that underwent disassembly as a consequence of its arrival to the ER determined as indicated in Materials and Methods. Values represent the mean ± SEM of 3 independent determinations. ****p < 0.0001, ***p < 0.001; n. s.: differences not statistically significant; two-tailed Student’s t-test. (C) Presence of typhoid toxin in the cytosolic fractions (after retro-translocation) in the indicated knockout cells. Cells were incubated with purified typhoid toxin and fractionated to examine the amount of typhoid toxin in the cytosolic fractions by western blot analysis. (D) Quantification of the relative amount of typhoid toxin in the cytosolic fraction. Values represent the mean ± SEM of three independent experiments. ***p < 0.001, and **p < 0.01; two-tailed Student’s t-test. (E). Toxicity of cytolethal distending toxin in defective cell lines. The parent wild type (WT) and the indicated knockout cell lines were treated with 5 μg of C. jejuni CDT for 48 hr and subjected to flow cytometric cell cycle analysis. Values are the mean ± SD of five independent experiments. ***p < 0.001, **p < 0.01; n. s.: differences not statistically significant; two-tailed Student’s t-test.