Expression of Pseudomonas aeruginosa toxin ExoS effectively induces apoptosis in host cells - PubMed (original) (raw)

Expression of Pseudomonas aeruginosa toxin ExoS effectively induces apoptosis in host cells

Jinghua Jia et al. Infect Immun. 2006 Dec.

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Invasive strains of P. aeruginosa are known to induce apoptosis at a high frequency in HeLa cells and in many other cell lines, a process that is dependent on the ADP-ribosylation (ADPRT) activity of a type III secreted protein ExoS. In our previous report, it was proposed that P. aeruginosa secreting ExoS, upon infection, shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2, leading ultimately to cytochrome c release and activation of caspases. In this study, we demonstrate that the expression of ExoS in HeLa cells by eukaryotic expression vector effectively caused apoptosis in an ADPRT activity-dependent manner, indicating that ExoS alone is sufficient to trigger apoptotic death of host cells independent of any other bacterial factors. By expressing an EGFP-ExoS fusion protein, we were able to directly correlate the death of HeLa cells with the presence of intracellular ExoS and further proved the dependence of this process on both JNK activation and mitochondrial proapoptotic event. The cellular pathway responsible for the ExoS-induced cytotoxicity appears to be well conserved, since the expression of the ADPRT-competent ExoS also induced rapid cell death in the Drosophila melanogaster S2 cell lines. The presented study not only highlights the ability of ExoS ADPRT to modulate host cell signaling, eventually leading to apoptosis, but also establishes ExoS as a valuable tool, in principle, for the elucidation of apoptosis mechanisms.

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Figures

FIG. 1.

Caspase-3-dependent cytotoxicity caused by transfection of the exoS expression plasmid. (A) Disappearance of GFP signal after cotransfection. HeLa cells were cotransfected with pGFP reporter plasmid and the exoS expression plasmids as indicated, with (▪) or without (□) the presence of 1 μM DEVD-CHO, a cell-permeable caspase-3 inhibitor. HeLa cells were observed under a fluorescence microscope using GFP fluorescence at 24 h posttransfection, and GFP-positive cells were quantified from five random views. Plasmids used for cotransfection with pGFP are indicated as follows: pcDNA4, vector control; pexoS, wild-type exoS in pcDNA4(pJJ0040); pexoSEA, exoS(E381A) in pcDNA4(pJJ0043); pexoSRK, exoS(R146K) in pcDNA4(pJJ0042); and pexoSRKEA, exoS(R146K/E381A) in pcDNA4(pJJ0044). As a solvent control, HeLa cells were treated with dimethyl sulfoxide (DEVD-CHO, 0 M). The data are means ± the SD of the counts of GFP-positive cells from three replicates. P values were calculated by comparing DEVD-CHO-treated (1 μM) and untreated (0 M) groups (***, P < 0.001). (B) Nuclear condensation caused by transfection with pcDNA4-exoS. HeLa cells were transfected with p_exoS_, wild-type exoS in pcDNA4, or pcDNA4 alone. HeLa cells were collected at 36 h posttransfection, stained with Hoechst dye, and subjected to fluorescence microscopy. Five fields were randomly sampled from each experimental population, and all of the cells stained with Hoechst dye in each field were counted up to 500 in total. The total number of apoptotic cells with condensed or fragmented nuclei was determined in the five sampled regions and was expressed as follows: percentage of apoptosis per sample = (number of apoptotic cells/total number of cells) × 100%. During and after transfection, HeLa cells were treated with the indicated amount of DEVD-CHO, a cell-permeable specific caspase-3 inhibitor. The data are means ± the SD of the percentages from three replicates. Significant differences between certain DEVD-CHO treated and untreated groups are indicated (**, P < 0.01). (C) Apoptosis after transfection with the indicated exoS expression constructs. In each experiment, the apoptotic nucleus percent ratio was calculated by comparing the calculated percentage of apoptosis to that of the pcDNA4. During and after transfection, HeLa cells were treated without (control) or with indicated amounts of DEVD-CHO or VAD-fmk, a cell-permeable specific caspase-3 or a pan-caspase inhibitor, respectively. TNF/CHX, treatment with TNF-α and CHX was used as a positive control for apoptosis induction. The data are means ± the SD of the ratios from three replicates. Significant differences between certain DEVD-CHO- or VAD-fmk-treated and untreated control groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG. 2.

GAP activity of ExoS is not essential for apoptosis induction by PAK. HeLa cells were infected with the indicated PAK derivative strains (multiplicity of infection of 20) or left uninfected (CTRL). At 5 h postinfection, cells were collected, lysed, and subjected to caspase-3 assay. The exoS and exoT double mutant PAK (ST) was complemented with vector (ST/V) or with exoS (ST/S), exoS(E381A) [ST/s(EA)], exoS(R146K) [ST/s(RK)], or exoS(R146K/E381A) [ST/s(RKEA)] on the same vector. The data are means ± the SD of the ratios from four replicates. Significant differences between ST/V (V) and other PAK-derived strains are indicated ([Image: see text] [Image: see text] , P < 0.01; [Image: see text] [Image: see text] [Image: see text] , P < 0.001).

FIG. 3.

Expression of EGFP fusion proteins in HeLa cell after transient transfection. Wild type and exoS, exoS(E381A), and exoS(R146K/E381A) mutants were cloned into pEGFP-C1, with the resulting fusion constructs labeled as follows: S, wild-type ExoS; EA, ExoSE318A; and RKEA, ExoSR146KE381A double mutant. At 12 h posttransfection, the total cellular proteins were collected. The expression of EGFP or fusion proteins was determined by Western blotting with antibodies to ExoS or GFP. Arrows point to the full-length fusion protein band; the arrowhead indicates the EGFP band. Equal protein loading was controlled by Ponceau S staining of membranes and Western blotting for β-actin.

FIG. 4.

(A) Transient expression of EGFP-ExoS in HeLa cells. HeLa cells were transfected with EGFP fusion protein constructs as indicated. HeLa cells were observed under a microscope using GFP fluorescence (A to D) or a bright-field (BF) filter (E to H) at 16 h posttransfection. A and E, EGFP vector control; B and F, EGFP-S, wild-type exoS in fusion construct; C and G, EGFP-EA, exoS(E381A) in fusion construct; D and H, EGFP-RKEA, exoS(R146K) in fusion construct. In each pair of images, the arrows refer to the same cells with detectable GFP. (B) FACS analysis of GFP signals in HeLa cells transiently transfected with fusion constructs. HeLa cells were collected at 24 h posttransfection and subjected to flow cytometry analysis using FL-1 channel on a FACSort cytometer. Each color-coded line on the histogram represents a collection of cells that were transfected with an indicated construct, with the GFP intensity of each cell on the x axis and counts of the cells with the same intensity on the y axis. A threshold intensity for a GFP-positive cell (GFP+ in the figure) was set based on a vector control, and the percentages for GFP-positive cells, of the total, are presented on the right. CTRL means no plasmid DNA was added for transfection. The results are from a single experiment that is representative of three replicated experiments. (C) Cytosolic localization of fusion proteins in HeLa cells after transient transfection with fusion protein expression plasmids. HeLa cells were transfected with EGFP fusion protein constructs as indicated. The resulting HeLa cells were fixed for 16 h after transfection and then stained with DAPI and observed under a phase-contrast DMIRB inverted fluorescence microscope (Leica, Germany) using a GFP fluorescence (a and b) or UV (DAPI, c and d) filter; merged images of GFP and DAPI are also shown (e and f). Panels a, c, and e show EGFP-EA [exoS(E381A)] and panels b, c, and f show EGFP-RKEA [exoS(R146K/E381A)] in fusion constructs.

FIG. 4.

FIG. 5.

(A) Caspase-3 activation in EGFP-ExoS transfected HeLa cells. HeLa cells were transfected with EGFP fusion protein constructs as indicated. The resulting HeLa cells were collected 24 h after transfection, and a caspase-3 activation assay was performed using Red-DEVD-FMK as described in Materials and Methods. Flow cytometry analysis was performed using a FL-2 channel on a FACSort cytometer. Dot density analysis of each sample is represented as the FL-2 intensity of a single cell (x axis) and the forward light scatter of the cells (y axis). The index for the gating population with activated caspase-3 in this assay was determined from the analysis pattern for HeLa cells treated with 2 μM STS for 4 h, which served as a positive control (subpanel B). Remaining subpanels: A, CTRL, no DNA was added for the transfection procedure; C, EGFP-S, wild-type exoS in fusion construct; D, EGFP-EA, exoS(E381A) in fusion construct; E, EGFP, vector control; F, EGFP-RKEA, exoS(R146K/E381A) in fusion construct. (B) Caspase-3 activation in total transfected HeLa cells is reduced by pan-caspase inhibitor. Caspase-3 activation in HeLa cells was assayed 24 h after transfection with the indicated constructs or 3 h after STS treatment. HeLa cells were treated with Boc-

-fmk at 80 μM immediately after transfection. The data are means ± the SD from three replicates from one transfection experiment. Significant differences between Boc-

-fmk-treated and untreated (CTRL) groups are shown (** P < 0.01).

FIG. 6.

Caspase-3 activation in EGFP-ExoS transfected HeLa cells is dependent on JNK1 activation and cytochrome c release. (A and B) HeLa/pcDNA3 (vector) or HeLa/DN JNK1 cells (A) and HeLa/Neo vector (vector) or HeLa/Bcl-xL cells (B) were transfected with the EGFP-ExoS construct and subjected to caspase-3 activation assay with Red-DEVD-FMK as described above. As a control for apoptosis induction, STS (2 μM) was used to treat the indicated cells for 3 h. The data are means ± the SD from three replicates from one transfection experiment. Significant differences between HeLa/pcDNA3 (vector) or HeLa/DN JNK1 cells (A) or between HeLa/Neo vector (vector) and HeLa/Bcl-xL cells (B) are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG. 7.

(A) ExoS induced cell death in Drosophila S2 cells. Similar to its effect on mammalian cells, the cytotoxicity of ExoS in Drosophila S2 cells is dependent on its ADP-ribosylating activity (EA, ExosE381A; RK, ExoSR146K; RKEA, ExoSR146K/E381A). Wild type and ExoSRK killed essentially 100% transfected cells, whereas the ExoSEA and ExoSRKEA double mutants have dramatically reduced cytotoxic activity. (B) While increasing the level of dIAP1 significantly suppressed reaper-induced cell death, it had little effect on ExoS-induced cytotoxicity. The colors of the bar reflect the relative ratio between proapoptotic gene constructs and Diap1. For both panels A and B, the data are represented as averages (n = 5 and 4, respectively), and error bars indicate the SD.

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Expression of Pseudomonas aeruginosa toxin ExoS effectively induces apoptosis in host cells - PubMed (original) (raw)