Fig 1.
ExoT/ADPRT-intoxicated HeLa cells exhibit anoikis markers.
(A) HeLa cells were infected with PA103∆exoU (∆U) at MOI ~10. Video images were captured every 15 min at 200× magnification. Video frames show a representative ExoT-intoxicated cell (yellow arrow) undergoing cell rounding at 1h and death, as identified by uptake of propidium iodide (red) at 9h. Cell tracking performed by ImageJ, shows this cell movement just prior to cell rounding up until cell death. (B) HeLa cells were treated with PBS (Mock) or infected with PA103∆exoU (∆U); PA103∆exoU/exoT(R149K) (∆U/T(G-A+)); or the T3SS mutant PA103 pscJ::Tn5 (pscJ) at MOI~10. At indicated time points after infection, cell lysates were probed for phosphorylated/activated forms of p38β or JNK (p-p38β or p-JNK) by Western blotting. The fold changes in expression, as compared to mock, are shown underneath. The data were normalized to GAPDH, which was used as loading control. (C and E) HeLa cells were transfected with expression vectors harboring wild type ExoT (pExoT), ExoT with functional ADPRT domain (pExoT(G-A+)), inactive ExoT (pExoT(G-A-)), or empty vector (pGFP). ~24 hr after transfection, cells were fixed and analyzed for either phospho-JNK (p-JNK) (C) or phospho-p38β (E) by IF microscopy. Representative images are shown in (C and E) and the respective expression levels were determined by densitometry and are shown as the mean fluorescent intensity (MFI) ± SEM in (D and F) (* Signifies p<0.001. One-way ANOVA compared with pGFP. Scale bar = 25μm). These data indicate that ExoT/ADPRT is both necessary and sufficient to activate JNK and p38β. (G) HeLa cells transiently transfected as described above and the transient transfection efficiencies were evaluated by Western blotting using anti-GFP antibody. GAPDH was used as a loading control. All data are representative of 3 independent experiments.
Fig 2.
ExoT/ADPRT interferes with integrin-mediated survival signaling.
(A) HeLa cells were treated with PBS or infected with PA103∆exoU (∆U); PA103∆exoU/exoT(R149K) (∆U/T(G-A+)); or the T3SS mutant PA103 pscJ::Tn5 (pscJ) at MOI~10. At indicated time points after infection, cell lysates were probed for activated Akt (p-AktS473), Akt-mediated GSK-3β inactivation by phosphorylation (p-GSK-3βS9), or β-catenin levels by Western blotting. The data were normalized to GAPDH and the fold changes in levels, as compared to mock, are shown underneath. (B) β-catenin transcriptional activity was assessed by transfecting HeLa cells with the TOPFlash luciferase reporter plasmid for 24 hr before infecting the cells with either ΔU, ΔU/T(G-A+), pscJ, or PBS. At indicated time points luciferase was assessed by a luminometer using the Luciferase Assay System (see Experimental Procedures). The experiment was performed in triplicate and the luciferase readings were normalized to baseline levels. Data are shown as mean ± SEM, * p<0.001, Student’s t-test).
Fig 3.
Anoikis resistant HeLa S3 cells are resistant to ExoT/ADPRT-induced cytotoxicity.
HeLa or HeLa S3 cells were treated with PBS (Mock) or infected with PA103∆exoU (∆U); PA103∆exoU/exoT(R149K) (∆U/T(G-A+)); or the T3SS mutant PA103 pscJ::Tn5 (pscJ) at MOI~10. Cytotoxicity in host cells was assessed by simultaneous phase and fluorescent time-lapse microscopy, using PI uptake (red cells are dead). Video images were captured every 15 min. selected movie frames at indicated time points are shown in (A, B) and the corresponding cytotoxicity assessment (evaluated by PI fluorescence intensity measurements for total positive pixels per frame) are shown in (C, D) respectively. Cytotoxicity is shown as the mean of 3 independent experiments. (E) ExoT level in infected HeLa and HeLa S3 cells after 4hr infection was determined by Western blotting. (Scale bar = 100 μm).
Fig 4.
CrkI/R38K mutant phenocopies ExoT/ADPRT-induced apoptosis in HeLa cells.
HeLa cells were transiently transfected with pIRES2-GFP expression vector harboring wild type CrkI (pCrkI), SH2 DN (pCrkI/R38K), all directly fused to GFP C-terminally, or empty vector (pGFP) in the absence (A) or presence (B) of Z-VAD pan-caspase inhibitor. PI was added to identify dying cells and cell death was analyzed by timelapse IF videomicroscopy. (C) The tabulated results, collected from multiple movies, are shown. (D) Transfection efficiencies were evaluated by Western blotting, using anti-GFP antibody and cells lysates from HeLa cells transfected as in (A). GAPDH was used as a loading control. The black arrowhead points to the position of GFP from the vector alone and the white arrowhead indicates CrkI-GFP and CrkI/R38K-GFP. (E) The time to death was defined as the time of expression of the transfected gene (appearance of green) to the time of PI uptake (appearance of yellow) and expressed as the mean ± SEM. Note that expression of SH2 DN CrkI induces potent apoptosis and kinetically phenocopies ExoT and ExoT/ADPRT-induced apoptosis. (* Signifies significance with p<0.01, χ2 analyses. Scale bar = 25 μm).
Fig 5.
Crk mediates ExoT-induced apoptosis.
Crk-/- cells were transiently transfected with pCrkI, or pGFP on 3 consecutive days to obtain more transfected cells. 24 hr after the final transfection, cells were infected with either ∆U, ∆U/T(G-A+), or pscJ at MOI~10. Cytotoxicity of transfected host cells (green) was assessed by fluorescent time-lapse microscopy, using PI uptake (red cells are dead). Cytotoxicity is expressed as a percentage of the total number of transfected cells. Video images were captured every 15 min and selected movie frames at indicated time points are shown in (A). (For clarity, phase panels were excluded from the merged images). The corresponding tabulated data are shown in (B). (C) The corresponding time to death, defined as the time of infection to the time of PI uptake (red) is expressed as the mean ± SEM. The data in B and C comprise 3 independent experiments. Statistical analysis was performed using one-way ANOVA.
Fig 6.
CrkI/R38K induces cytotoxicity in Crk-/- cells.
Crk-/- cells were transiently transfected with pCrkI, pCrkI/R38K, pCrkI/R38K, W170K or the vector control pGFP. Video images were captured every 15 min and selected movie frames at indicated time points are shown in (A) (For clarity, phase panels were excluded from the merged images). (B) Cytotoxicity of transfected host cells (green) was assessed by fluorescent time-lapse microscopy, using PI uptake (red cells are dead). Cytotoxicity is expressed as a percentage of the total number of transfected cells. * Signifies significance with p<0.001 by χ2 analyses analysis. Data are representative of 3 independent experiments.
Fig 7.
ExoT and ADPRT disrupt focal adhesion sites.
(A-D) HeLa cells were treated with PBS or infected with ∆U, ∆U/T(G-A+), or pscJ at MOI~10. Four hours after infection, cells were fixed and stained with nuclear stain DAPI and p-FAK (A) or p-p130Cas (B). Representative images are shown in (A and B) and the tabulated results are shown in (C and D). (E-H) HeLa cells were transiently transfected with pIRES2-GFP expression vector harboring ExoT (pExoT), ExoT/ADPRT (pExoT(G-A+), or inactive ExoT (pExoT(G-A-), all C-terminally fused to GFP, or empty vector (pGFP). 24 hr after transfection, cells were fixed and stained with nuclear stain DAPI and analyzed for p-FAK and p-p130Cas localization to the FA sites. Representative images are shown in (E and F) and the tabulated results from 3 independent experiments are shown in (G and H). (Statistical analysis was performed using one-way ANOVA. Scale bar = 25 μm). (I) HeLa cells were infected as in (A). 4hr after infection, the cell lysates were probed for total FAK and p130Cas or their phosphorylated forms (p-FAKY397 & p-p130CasY165 respectively) by Western blotting. Expression levels were normalized to GAPDH, which was used as the loading control.
Fig 8.
CrkI/R38K mutant disrupts FA sites in HeLa and Crk-/- cells, phenocopying ExoT/ADPRT adverse effect on FA sites.
HeLa cells were transiently transfected with pIRES2-GFP expression vector harboring wild-type CrkI (pCrkI), SH2 DN (pCrkI/R38K), all C-terminally fused to GFP, or empty vector (pGFP). 24 hr after transfection, cells were fixed and stained with nuclear stain DAPI and analyzed for p-FAK localization to the FA sites. Representative images are shown in (A and D) and the tabulated results from 3 independent experiments are shown in (B-C and E-F). (E) Crk-/- cells were transiently transfected with pIRES2-GFP expression vector harboring wild-type CrkI (pCrkI), SH2 DN (pCrkI/R38K), or SH2 and SH3 double mutant (pCrkI/R38K,W170K), all C-terminally fused to GFP, or empty vector (pGFP). 24 hr after transfection, cells were fixed and stained with nuclear stain DAPI and analyzed for p-FAK localization to the FA sites. Representative images and enlargements of fields indicated by a white box are shown in (G) and the tabulated results from 3 independent experiments are shown in (H-I). (Statistical analysis was performed using one-way ANOVA. Scale bar = 25 μm).