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Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo.

Lembo-Fazio L, Nigro G, Noël G, Rossi G, Chiara F, Tsilingiri K, Rescigno M, Rasola A, Bernardini ML - Cell Death Dis (2011)

Bottom Line: Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues.However, they are equally able to protect host cells from death.To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Biotecnologie Charles Darwin, Sapienza-Università di Roma, Roma, Italy.

ABSTRACT
Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens.

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Shigella-infected HeLa cells undergo intrinsic apoptosis. (a) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. (b) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In (a and b), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H2O2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with siRNA for caspase-9 (c) or for caspase-8 (d) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF-α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test
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fig3: Shigella-infected HeLa cells undergo intrinsic apoptosis. (a) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. (b) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In (a and b), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H2O2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with siRNA for caspase-9 (c) or for caspase-8 (d) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF-α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test

Mentions: To understand the relative role played by each of the two apical caspases in caspase-3 activation, previously determined by luminometric assay, we carried out a dual strategy. First, we assessed whether Shigella infection induced an early mitochondrial depolarization, which triggers caspase-9-mediated activation of caspase-3 in intrinsic apoptotic pathway. Other groups have already reported mitochondrial dysfunction on Shigella infection of fibroblasts.12, 16 Accordingly, by using flow cytometry analysis we found that about one-third of the infected cells displayed depolarized mitochondria as early as 1 h p.i. (Figures 3a and b). In addition, we observed that, starting from the first hour of infection, a fraction of cells undergoing mitochondrial membrane depolarization also displayed the caspase-3 activation, as assayed by the binding of the fluorescent-coupled DEVD inhibitor to the activated form of caspase-3 (FLICA caspase-3 (FAM-DEVD-FMK)). Second, we exploited an RNAi strategy in order to silence either caspase-8 or caspase-9. We observed that the caspase-3 activity was significantly reduced in the presence of RNAi for caspase-9, that is, 1.7±0.19 versus 3.9±0.05 at 3 h of incubation p.i. and 1.4±0.3 versus 6±0.19 following 5 h of incubation p.i. (Figure 3c). We did not observe any difference in caspase-3 values in the presence of RNAi for caspase-8 (Figure 3d), suggesting that an early caspase-8 activation is not necessary to induce apoptosis following Shigella infection, whereas the intrinsic apoptotic pathway is responsible for effector caspase activation.


Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo.

Lembo-Fazio L, Nigro G, Noël G, Rossi G, Chiara F, Tsilingiri K, Rescigno M, Rasola A, Bernardini ML - Cell Death Dis (2011)

Shigella-infected HeLa cells undergo intrinsic apoptosis. (a) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. (b) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In (a and b), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H2O2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with siRNA for caspase-9 (c) or for caspase-8 (d) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF-α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig3: Shigella-infected HeLa cells undergo intrinsic apoptosis. (a) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. (b) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In (a and b), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H2O2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with siRNA for caspase-9 (c) or for caspase-8 (d) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF-α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test
Mentions: To understand the relative role played by each of the two apical caspases in caspase-3 activation, previously determined by luminometric assay, we carried out a dual strategy. First, we assessed whether Shigella infection induced an early mitochondrial depolarization, which triggers caspase-9-mediated activation of caspase-3 in intrinsic apoptotic pathway. Other groups have already reported mitochondrial dysfunction on Shigella infection of fibroblasts.12, 16 Accordingly, by using flow cytometry analysis we found that about one-third of the infected cells displayed depolarized mitochondria as early as 1 h p.i. (Figures 3a and b). In addition, we observed that, starting from the first hour of infection, a fraction of cells undergoing mitochondrial membrane depolarization also displayed the caspase-3 activation, as assayed by the binding of the fluorescent-coupled DEVD inhibitor to the activated form of caspase-3 (FLICA caspase-3 (FAM-DEVD-FMK)). Second, we exploited an RNAi strategy in order to silence either caspase-8 or caspase-9. We observed that the caspase-3 activity was significantly reduced in the presence of RNAi for caspase-9, that is, 1.7±0.19 versus 3.9±0.05 at 3 h of incubation p.i. and 1.4±0.3 versus 6±0.19 following 5 h of incubation p.i. (Figure 3c). We did not observe any difference in caspase-3 values in the presence of RNAi for caspase-8 (Figure 3d), suggesting that an early caspase-8 activation is not necessary to induce apoptosis following Shigella infection, whereas the intrinsic apoptotic pathway is responsible for effector caspase activation.

Bottom Line: Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues.However, they are equally able to protect host cells from death.To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Biotecnologie Charles Darwin, Sapienza-Università di Roma, Roma, Italy.

ABSTRACT
Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens.

Show MeSH
Related in: MedlinePlus