Fig 1.
Typhoid toxin traffics to the ER by retrograde transport.
(A) Tracking typhoid toxin (TT) transport by immunofluorescence microscopy. HEK293T cells were incubated with Oregon Green-488-labeled typhoid toxin (green) at 4°C for 30 min, washed, and fixed with 4% paraformaldehyde (binding). Alternatively, after 30 min incubation at 4°C, cells were washed, and then switched to 37°C, incubated for 0.5, 2, and 8 hs and fixed as indicated above. Fixed cells were stained with an anti-GM130 antibody (red) and visualized by fluorescence microscopy. Scale bar, 5 μm. (B and C) Typhoid toxin undergoes retrograde transport to the trans-Golgi network (TGN). (B) Schematic representation of the assay to detect typhoid toxin transport through the Golgi. (C) HEK293T cells transiently expressing myc epitope tagged SNAP-Galactosyl transferase 1 (Myc-SNAP-GalT) were treated with BG-NHS-labeled (BG-TT) or unlabeled (TT) typhoid toxin for 6 hr at 37°C. BG-labeled toxin molecules that were “captured” by SNAP-GalT formed chimeric protein complexes (indicated as "TT-SNAP-GalT") that were detected by Western blot analysis with an antibody directed to the Myc epitope. Dotted lines indicate places where the experimentally relevant lanes were spliced together (all lanes originate from a single gel). (D and E) Typhoid toxin transport to the endoplasmic reticulum (ER). (D) Schematic representation of the typhoid toxin-disassembly assay in the ER. (E) HEK293T cells were treated with purified typhoid toxin for 30 min at 37°C and lysed at the indicated time points. The mobility of typhoid toxin in SDS-PAGE in the presence or absence of DTT (as indicated) was then analyzed by Western blot with an antibody to CdtB. The positions of CdtB and the CdtB-PltA heteromeric complex are indicated. * denotes the migration of a non-specific cross-reacting protein (F) Typhoid toxin retro-translocation from the ER to the cell cytosol. HEK293T cells were incubated with purified typhoid toxin at 37°C and then harvested at the indicated time points. Cells were selectively permeabilized with digitonin and the presence of typhoid toxin in the cytosolic fraction was detected by Western blot analysis with an antibody to CdtB.
Fig 2.
A genome-wide CRISPR/Cas9-mediated gene inactivation screen identifies genes essential for typhoid toxin intoxication.
(A) A schematic of the workflow for the screen to identify genes involved in typhoid toxin toxicity. HEK293T cells expressing Cas9 were transduced with a lentiviral library encoding sgRNA targeting human genes as described in Material and Methods. After puromycin selection, cells were split and either mock treated or treated with typhoid toxin. Cells that survived the toxin treatment or that were mock treated were subjected to nucleotide sequence analysis as indicated in Materials and Methods. Sequences were aligned to the reference genome and high-quality reads were analyzed with the MAGeCK algorithm to identify positively-selected genes. (B and C) Scatter plots of effect size (log fold change; x axis) versus P value (-log10 raw P-value; y-axis) for all genes. Three replicates were performed for each sub-library and the MAGeCK algorithm was used to compare treated with untreated cells across replicates for the human GeCKO v.2 sub-library A and B as described in Material and Methods. Inactivation of the genes colored in red conferred resistance to typhoid toxin with a P-value cutoff corresponding to 15% FDR. (D) Gene ontology term enrichment analysis of genes whose inactivation conferred toxin resistance. The P-values represent the probability of the identified genes to be annotated to a particular GO term relative to all the annotated human genes. GO terms are shown depicting biological processes in black and cellular components in gray.
Fig 3.
Validation of candidate genes involved in typhoid toxin intoxication.
(A) Genotyping of the CRISPR/Cas9-generated knockout cells. Genomic DNA was purified from CRISPR-Cas9 edited HEK293T cell lines and analyzed by PCR with specific primers listed in S3 Table. The TMED2-deficient cell line was examined by Western blot with an anti TEMED2-specific antibody. Dotted lines indicate places where the experimentally relevant lanes were spliced together (all lanes originate from a single gel). (B) Relative toxicity of typhoid toxin in the indicated knockout cell lines after treatment with a serial dilution of purified toxin. The relative toxicity was determined from the percentage of cells at the G2M phase fitted by nonlinear regression as indicated in the Materials and Methods. Values, which were normalized relative to wild type (considered 100) are the mean ± SEM of independent determinations. ****p < 0.0001; n. s.: differences not statistically significant; two-tailed Student’s t-test.
Fig 4.
Characterization of typhoid toxin trafficking to the TGN in CRISPR/Cas9-edited cell lines.
(A and B) Co-localization of typhoid toxin with the Golgi marker GM130. (A) Wild-type and the indicated knockout cell lines were treated with Oregon-488 labeled typhoid toxin (green) and 2 hours after toxin treatment, cells were stained with an anti-GM130 antibody (red) as described in Material and Methods. Scale bar, 5 μm. (B) The co-localization between typhoid toxin and GM130 was determined as described in Material and Methods. Values represent the relative co-localization (normalized to wild type) and are the mean ± SEM of three independent experiments. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05; two-tailed Student’s t-test. (C and D) Typhoid toxin Golgi localization determined by SNAP-capture. (C) Cells expressing Myc-epitope tagged SNAP-GalT (Myc-SNAP-GalT) were incubated with BG-labeled typhoid toxin for 6 hr and subsequently analyzed by Western blot with an anti-Myc antibody to detect typhoid toxin/SNAP-GalT chimeric protein complexes (TT-SNAP-GalT) and anti β-actin antibody as a loading control. The migration position of the uncomplexed (Myc-SNAP-GalT) and toxin complexed GalT-SNAP (TT-SNAP-GalT) are indicated. (D) Relative amounts of TT-SNAP-GalT quantified from the blots as indicated in Materials and Methods. Values represent the relative intensity of all bands associated with TT-SNAP-GalT (normalized for loading and relative to the values of wild type, which were considered 100) and are the mean ± SEM of 3 independent determinations. ****p < 0.0001; ***p < 0.001; *p < 0.05; n. s.: differences not statistically significant; two-tailed Student’s t-test.
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.
Fig 6.
Common and distinct toxin transport pathways revealed by genome wide screens.
Boxes indicate known physical complexes. Proteins involved in the transport of all the indicated toxins are depicted in yellow while proteins uniquely involved in typhoid toxin transport are indicated in blue. The trafficking models of cholera toxin and ricin are summarized from Gilbert’s et al. [50] and Tian et al. [51], respectively.