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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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Gadd45α triggers Shigella-mediated apoptotic cell death in infected HeLa cells. Activity of caspase-8 (A), caspase-9 (B) and caspase-3 (C), and TUNEL assay (E and F) on HeLa cells transiently transfected with a Gadd45α or a scramble siRNA. Cells were infected with M90T at MOI of 100 for the reported time points. HeLa cells treated with STP or with CHX plus TNF-α, as detailed in Figure 2, were used as a control (D). HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test. (F) A representative cytofluorimetric output of TUNEL analysis performed at 1 h (upper panel, a and b), 3 h (middle panel, c and d) and 5 h of incubation p.i. (lower panel, e and f)
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fig5: Gadd45α triggers Shigella-mediated apoptotic cell death in infected HeLa cells. Activity of caspase-8 (A), caspase-9 (B) and caspase-3 (C), and TUNEL assay (E and F) on HeLa cells transiently transfected with a Gadd45α or a scramble siRNA. Cells were infected with M90T at MOI of 100 for the reported time points. HeLa cells treated with STP or with CHX plus TNF-α, as detailed in Figure 2, were used as a control (D). HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test. (F) A representative cytofluorimetric output of TUNEL analysis performed at 1 h (upper panel, a and b), 3 h (middle panel, c and d) and 5 h of incubation p.i. (lower panel, e and f)

Mentions: Gadd45α is a stress-inducible gene regulated by a variety of genotoxic and non-genotoxic stresses. Gadd45α may have an important role as both a pro-apoptotic17, 18, 19 and pro-survival factor as well.20 Given the sustained upregulation of Gadd45α in infected HeLa cells, we asked whether and to which extent Gadd45α could be involved in Shigella-mediated apoptosis. We analyzed caspase-9, -8 and -3 activity and DNA fragmentation in HeLa cells depleted of Gadd45α through RNAi and infected with M90T as above. Uninfected cells or cells transfected with scramble siRNA and infected as above were used as controls. We found that caspase-9 maturation was dramatically abrogated in the presence of RNAi for Gadd45α, starting from 1 h of incubation p.i. (Figure 5B), while caspase-8 activity remained unaltered (Figure 5A).


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)

Gadd45α triggers Shigella-mediated apoptotic cell death in infected HeLa cells. Activity of caspase-8 (A), caspase-9 (B) and caspase-3 (C), and TUNEL assay (E and F) on HeLa cells transiently transfected with a Gadd45α or a scramble siRNA. Cells were infected with M90T at MOI of 100 for the reported time points. HeLa cells treated with STP or with CHX plus TNF-α, as detailed in Figure 2, were used as a control (D). HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test. (F) A representative cytofluorimetric output of TUNEL analysis performed at 1 h (upper panel, a and b), 3 h (middle panel, c and d) and 5 h of incubation p.i. (lower panel, e and f)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3101704&req=5

fig5: Gadd45α triggers Shigella-mediated apoptotic cell death in infected HeLa cells. Activity of caspase-8 (A), caspase-9 (B) and caspase-3 (C), and TUNEL assay (E and F) on HeLa cells transiently transfected with a Gadd45α or a scramble siRNA. Cells were infected with M90T at MOI of 100 for the reported time points. HeLa cells treated with STP or with CHX plus TNF-α, as detailed in Figure 2, were used as a control (D). HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. *P<0.05, **P<0.01, ***P<0.001 after Student's t-test. (F) A representative cytofluorimetric output of TUNEL analysis performed at 1 h (upper panel, a and b), 3 h (middle panel, c and d) and 5 h of incubation p.i. (lower panel, e and f)
Mentions: Gadd45α is a stress-inducible gene regulated by a variety of genotoxic and non-genotoxic stresses. Gadd45α may have an important role as both a pro-apoptotic17, 18, 19 and pro-survival factor as well.20 Given the sustained upregulation of Gadd45α in infected HeLa cells, we asked whether and to which extent Gadd45α could be involved in Shigella-mediated apoptosis. We analyzed caspase-9, -8 and -3 activity and DNA fragmentation in HeLa cells depleted of Gadd45α through RNAi and infected with M90T as above. Uninfected cells or cells transfected with scramble siRNA and infected as above were used as controls. We found that caspase-9 maturation was dramatically abrogated in the presence of RNAi for Gadd45α, starting from 1 h of incubation p.i. (Figure 5B), while caspase-8 activity remained unaltered (Figure 5A).

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