doi: 10.1128/mBio.00479-16.
EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage
Affiliations
- PMID: 27329750
- PMCID: PMC4916375
- DOI: 10.1128/mBio.00479-16
Item in Clipboard
EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage
Antonio Serapio-Palacios et al.
mBio.
.
Display options
Format
Display options
Format
Abstract
Enteropathogenic Escherichia coli (EPEC) has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS), but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK), which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i) a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii) translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii) cytochrome c release from mitochondria to the cytoplasm, (iv) loss of mitochondrial membrane potential, (v) caspase-9 activation, (vi) cleavage of procaspase-3 and (vii) an increase in caspase-3 activity, (viii) PARP proteolysis, and (ix) nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC.
Importance: EspC, an autotransporter protein with serine protease activity, has cytotoxic effects on epithelial cells during EPEC infection. EspC causes cytotoxicity by cleaving fodrin, a cytoskeletal actin-associated protein, and focal adhesion proteins (i.e., FAK); interestingly, these proteins are also cleaved during apoptosis and necrosis. Here we show that EspC is able to cause cell death, which is characterized by apoptosis: by dissecting the apoptotic pathway and considering that EspC is translocated by an injectisome, we found that EspC induces the mitochondrial apoptotic pathway. Remarkably, EspC activates this pathway by two distinct mechanisms-either by using or not using its serine protease motif. Thus, we show for the first time that this serine protease motif is able to cleave procaspase-3, thereby reaching the terminal stages of caspase cascade activation leading to apoptosis. Furthermore, this overlapped apoptosis appears to potentiate cell death through necrosis, where EspC induces calpain activation and increases intracellular calcium.
Copyright © 2016 Serapio-Palacios and Navarro-Garcia.
Figures
EspC produces cell death on epithelial cells. (A to F) Cytotoxic effects induced by EspC-producing EPEC. Untreated HEp-2 cells were used as the control (A), and HEp-2 cells were infected with the EPEC wild-type (WT) strain (E2348/69) (B), ΔespC mutant (C), ΔespC/pespC complemented strain (D), ΔespC/pespCS256I complemented strain (E), or ΔescN mutant (F) for 4 h. Infected cells were fixed and permeabilized. Cells were immunostained with anti-EspC antibody, followed by a secondary antibody conjugated to fluorescein isothiocyanate (FITC), and the actin cytoskeleton was detected with rhodamine-phalloidin. Slides were observed using a Leica TCS SP8 confocal microscope. Scale bar, 20 μm. (G) Cell death induced by EspC-producing EPEC. HEp-2 cells were infected with the EPEC WT, ΔespC, ΔespC/pespC, ΔespC/pespCS256I, or ΔescN strain at an MOI of 10 for different lengths of time. Cells were harvested and stained with propidium iodide (PI) to perform a PI exclusion assay by flow cytometry. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P < 0.05; ***, P < 0.001).
EspC induces apoptosis and necrosis on epithelial cells. (A) Analyses of apoptotic and necrotic cells during kinetics of infection. FACS analysis via annexin V and PI staining was used to observe the induction of apoptosis and necrosis of HEp-2 cells infected by the EPEC WT, ΔespC, ΔespC/pespC, ΔespC/pespCS256I, or ΔescN strain at an MOI of 10 for the indicated lengths of time. Mock-infected cells were used as a negative control. Annexin V-negative and PI-negative cells represent live cells. Annexin V-positive and PI-negative cells represent the early apoptotic populations. Annexin V-positive and PI-positive cells represent the late apoptotic populations. Annexin V-negative and PI-positive cells represent the necrotic populations. Representative flow cytometric dot plots are shown. (B) Distribution of the infected cell populations at 4 h of infection. HEp-2 cells were infected with the bacterial strains for 4 h as indicated. Mock-infected cells were used as a negative control. Staurosporine (STS) at 1 μM and 0.1% Triton X-100 were used as positive controls for apoptosis or necrosis, respectively. The results were plotted and are shown as the mean ± SEM from at least 3 independent experiments.
EspC is required for caspase-3 activation and activity. (A) EspC-producing EPEC induces procaspase-3 cleavage. HEp-2 cells were infected with the indicated bacterial strains at an MOI of 10 at the different times indicated. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-caspase-3, anti-β-actin, and anti-EspC as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Samples were normalized using the maximum densitometric value. (B) Induction of procaspase-3 cleavage is complemented in the mutant strains by either espC or espF, and this effect is higher by espC complementation. Activation of caspase-3 was performed as indicated above, in cells infected by the indicated strains at 4 h of infection. (C) EspC-producing EPEC induces caspase-3 activity. Activity of caspase-3 was determined in HEp-2 cells infected by the EPEC WT, ΔespC, ΔespC/pespC, or ΔescN strain at an MOI of 10 for 4 h. The whole-cell lysates were subjected to the caspase activity assay by using a synthetic substrate as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (A) unpaired t test (*, P < 0.05; ***, P < 0.001) or (B and C) one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
EspC preferentially induces caspase-9 activity. (A) Activity of caspase-8 and (B) caspase-9 induced by EspC. HEp-2 cells were infected with the EPEC WT, ΔespC, ΔespC/pespC, or ΔescN strain at an MOI of 10 for 4 h. The whole-cell lysates were subjected to caspase activity assay by using specific substrates for each caspase as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P < 0.05; **, P < 0.01).
EspC induces a decrease in Bcl-2 protein levels. (A) Kinetics of detection of Bcl-2 in cells infected with the EPEC WT and ΔespC mutant. HEp-2 cells were infected with bacterial strains as indicated and for the indicated lengths of time. The asterisk indicates a clear Bcl-2 decrease induced by EPEC (B) detection of Bcl-2 at 4 h of infection with the EPEC WT, ΔespC, ΔespC/pespC, ΔespC/pespCS256I, or ΔescN strain at an MOI of 10. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-Bcl-2 and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Densitometries of three immunoblots were plotted. Results are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison with mock-infected cells (**, P < 0.01). (C) EspC is unable to cleave Bcl-2. Uninfected HEp-2 cells were lysed, and protein extracts were incubated with purified EspC or EspCS256I for 15, 30, and 60 min. Reactions were stopped with Laemmli buffer, and products were separated by SDS-PAGE, transferred to a PVDF membrane, and analyzed by immunoblotting using anti-Bcl-2, anti-β-actin, and anti-EspC as primary antibodies and HRP-conjugated anti-isotype secondary antibody.
EspC stimulates Bax translocation to mitochondria and cytochrome c release. EspC induces Bax translocation at 4 h of infection (A) and release of cytochrome c from mitochondria to cytosol (B). HEp-2 cells were infected with the different strains as indicated at an MOI of 10 and for the indicated lengths of time. Host cell cytosolic and mitochondrial fractions were separated and analyzed by immunoblotting using (A) anti-Bax or (B) anti-cytochrome c as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Blots were also probed for COX IV as a control marker for the mitochondrial fractions and GAPDH as a control marker for the cytosolic fractions. The blots shown are representative of three independent experiments. Asterisks indicate a clear increase induced by EPEC.
EspC induces depolarization of mitochondrial inner membrane (ΔΨm). (A) EspC is involved in the loss of mitochondrial membrane potential. HEp-2 cells were prestained with rhodamine 123 and then infected with bacterial strains as indicated at an MOI of 10 for 4 h. Purified EspC, EspCS256I, or EspC (900 nM) preincubated with PMSF was added during the infection to complement the double mutant as indicated. Staurosporine (STS) and Triton X-100 were used as positive controls. All cells were analyzed for the loss of mitochondrial inner membrane potential (ΔΨm) by flow cytometry. (B) Complementation of the EPEC ΔespC ΔespF strain with exogenous EspC. HEp-2 cells were infected with bacterial strains as indicated and supplemented with increasing molarities of purified EspC at an MOI of 10 for 3 h. Cells were processed and analyzed as indicated above. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the (A) WT or (B) ΔespC ΔespF strain (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
EspC induces PARP proteolysis and DNA nuclear fragmentation. (A) EspC induces PARP cleavage. HEp-2 cells were infected with the bacterial strains as indicated at an MOI of 10 and for the indicated times. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-cleaved PARP and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. (B) EspC induces DNA nuclear fragmentation. HEp-2 cells were infected with the EPEC WT, ΔespC, ΔespC/pespC, ΔespC/pespCS256I, or ΔescN strain at an MOI of 10 for 4 h. The cell cycle was measured by using propidium iodide (PI) staining; results are presented as histograms. (C) EspC induces an increase in the sub-G1 cell population. Cell distribution was measured by fluorescence intensity in the sub-G1 phase. Results are shown as percentages of the sub-G1 population. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using unpaired t test (*, P < 0.05) and one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (***, P < 0.001).
EspC also induces caspase-independent cell death. (A and C) Caspase inhibition does not block apoptosis induced by EPEC. HEp-2 cells were pretreated with 50 μM z-VAD-fmk or only DMSO for 1 h. Cells were infected with the EPEC WT, ΔespC, or ΔescN strain at an MOI of 10 for 4 h (A) or treated with 100 μM cisplatin (CDDP) for 18 h (C). Mock-infected cells were used as a negative control. Cells were harvested and labeled with annexin V and analyzed by flow cytometry. Annexin V-positive cells were considered apoptotic cells. (B and D) Caspase inhibition blocks caspase-3 activity induced by EPEC. Activity of caspase-3 was determined in HEp-2 cells pretreated with 50 μM z-VAD-fmk or only DMSO for 1 h and infected with the EPEC WT or ΔespC strain at an MOI of 10 for 4 h (B) or 100 μM CDDP for 18 h (D). The whole-cell lysates were subjected to the caspase-3 activity assay as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (*, P < 0.05; ***, P < 0.001).
Procaspase-3 is directly cleaved by EspC. (A) The presence of a caspase inhibitor plus a mutation in the serine protease motif causes a dramatic decrease in caspase-3 activation. HEp-2 cells were pretreated with 50 μM z-VAD-fmk or only DMSO for 1 h. Cells were infected with the EPEC WT, ΔespC, or ΔespC/pespCS256I strain at an MOI of 10 for 4 h. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-caspase-3 and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (**, P < 0.01). (B) Purified EspC cleaves procaspase-3. Uninfected HEp-2 cells were lysed, and protein extracts were incubated with the primary antibody anti-caspase-3 plus protein A-agarose overnight. Immunoprecipitated procaspase-3 was incubated with purified EspC or EspCS256I for 15, 30, and 60 min. Reactions were stopped with Laemmli buffer, and products were separated by SDS-PAGE, transferred to a PVDF membrane, and analyzed by immunoblotting using anti-caspase-3 as a primary antibody and HRP-conjugated anti-isotype secondary antibody. Heavy chains of antibodies used for the immunoprecipitation were used as a loading control. (C) Differential cleavage of procaspase-3 by EspC and caspases. HEp-2 cells were infected with the EPEC WT at an MOI of 10 for 4 h or treated with 100 μM cisplatin (CDDP) for 18 h and were processed as indicated above. Additionally immunoprecipitated procaspase-3 was incubated with purified EspC as indicated above. All samples were analyzed by immunoblotting using anti-caspase-3, anti-EspC, and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Asterisks indicate fragments of about 17 kDa, and dots indicate fragments of about 18 kDa.
EspC induces necrosis through calpain activation. (A) Kinetics of necrosis induction by the EPEC WT and ΔespC mutant and their inhibition by a calpain inhibitor. HEp-2 cells were pretreated with 50 μM calpain inhibitor I for 1 h or untreated. Cells were infected with either the EPEC WT or ΔespC mutant at an MOI of 10 for the indicated times. Cells were harvested, labeled with annexin V/PI, and analyzed by flow cytometry. Annexin V-negative and PI-positive cells were considered necrotic cells. (B) Comparison of cells infected with the different bacterial strains for 4 h. Cells were infected with the indicated strains, processed, and analyzed as indicated above. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (A) unpaired t test (**, P < 0.01; ***, P < 0.001) or (B) two-way ANOVA followed by Bonferroni’s multiple comparison test (*, P < 0.05; ***, P < 0.001).
EspC induces calpain activity during EPEC infection. (A) Measurement of calpain activity induced by EspC. HEp-2 cells were pretreated with 20 μM fluorescent substrate t-BOC-Leu-Met-CMAC upon cleavage by calpain. Cells were infected with the EPEC WT (E2348/69) or ΔespC mutant at an MOI of 10 for 4 h. Calpain activity was measured as increase in fluorescence intensity. (B) Kinetics of calpain activity induced by the EPEC WT or ΔespC mutant. Cells were infected with the bacterial strains at an MOI of 10 for the indicated times. Calpain activity was measured as increase of mean fluorescence intensity. All measurements were expressed relative to the calpain activity measured in mock-infected cells. Activity is represented as fold change relative to uninfected cells. (C) EspC causes an increase in calpain activity. HEp-2 cells were infected with the different bacterial strains as indicated at an MOI of 10 for 3 h. MDL28170, a specific calpain inhibitor, was included to demonstrate that the increase of fluorescence is specific to calpain-like proteases. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (B) unpaired t test (*, P < 0.05; **, P < 0.01) or (C) one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P < 0.05; **, P < 0.01).
EspC induces an increase in intracellular Ca2+ associated with necrosis. (A) Intracellular calcium chelation causes a decrease in the necrosis induced by EspC. HEp-2 cells were pretreated with 20 μM BAPTA-AM or only DMSO for 1 h. HEp-2 cells were infected with the indicated bacterial strains at an MOI of 10 for 4 h. Cells were harvested and labeled with annexin V and PI and analyzed by flow cytometry. Annexin V-negative and PI-positive cells were considered necrotic cells. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (*, P < 0.05; ***, P < 0.001). (B) Kinetics of increase of intracellular calcium induced by the EPEC WT and ΔespC strains. HEp-2 cells were pretreated with 4 μM Fluo-4 AM for 1 h. Cells were infected with either the EPEC WT or ΔespC mutant for the indicated times. Cells were harvested, and the mean Fluo-4 AM fluorescence intensity of each condition was analyzed by flow cytometry. (C) The presence of EspC increases intracellular calcium. HEp-2 cells were treated with the indicated bacterial strains for 4 h. BAPTA-AM, an intracellular calcium chelator, was used as a negative control. Cells were pretreated and treated as indicated above. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (B) unpaired t test (*, P < 0.05; **, P < 0.01) or (C) one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P < 0.05; ***, P < 0.001).
References
-
- Elliott SJ, Krejany EO, Mellies JL, Robins-Browne RM, Sasakawa C, Kaper JB. 2001. EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri. Infect Immun 69:4027–4033. doi:10.1128/IAI.69.6.4027-4033.2001. - DOI - PMC - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous