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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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Related in: MedlinePlus

Apoptosis assessment and Gadd45α expression in a human ex vivo organ culture (EVOC) invasion assay with S. flexneri M90T. (A) Dot blot of the distribution of LPS, Gadd45α, TUNEL and caspase-3 immunostained epithelial cells in sections of human colon biopsies infected with M90T or BS176 (12 h) or uninfected. Immunohistochemically stained cells were counted at × 400 magnification. For each sample, five view fields in five sections were considered for cell enumeration. Circles represent the mean values per sample while the horizontal line represents the mean value per sample category. ***P<0.001 after Student's t-test. (B) Histopathological and IH characterization of human colon sections infected with M90T or BS176 or uninfected. Upper panels (HE and LPS): HE (a, b, c) HE staining; LPS (d, e, f): IH staining of S. flexneri 5 LPS, in e and f bacterial LPS are indicated by arrows. In HE original magnification 200; in LPS original magnification × 400. Lower panels: analysis in serial sections of EVOC of immunolabeled mature caspase-3 (g, h, i), Gadd45α (j, k, l) and colocalization of Gadd45α immunostaining and positive TUNEL nuclei, Gadd45α/TUNEL (m, n, o). All relevant informations are reported in the figure. Arrowheads indicate TUNEL-positive cells and arrows indicate cell positively immunostained by the Gadd45α mAb. In caspase-3, Gadd45α and Gadd45α/TUNEL panels, original magnification × 400. Human colon NI: uninfected human colon; M90T-infected: EVOC infected over-night with 108 CFU of M90T; BS176-infected: EVOC infected over-night with 108 CFU of BS176
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fig6: Apoptosis assessment and Gadd45α expression in a human ex vivo organ culture (EVOC) invasion assay with S. flexneri M90T. (A) Dot blot of the distribution of LPS, Gadd45α, TUNEL and caspase-3 immunostained epithelial cells in sections of human colon biopsies infected with M90T or BS176 (12 h) or uninfected. Immunohistochemically stained cells were counted at × 400 magnification. For each sample, five view fields in five sections were considered for cell enumeration. Circles represent the mean values per sample while the horizontal line represents the mean value per sample category. ***P<0.001 after Student's t-test. (B) Histopathological and IH characterization of human colon sections infected with M90T or BS176 or uninfected. Upper panels (HE and LPS): HE (a, b, c) HE staining; LPS (d, e, f): IH staining of S. flexneri 5 LPS, in e and f bacterial LPS are indicated by arrows. In HE original magnification 200; in LPS original magnification × 400. Lower panels: analysis in serial sections of EVOC of immunolabeled mature caspase-3 (g, h, i), Gadd45α (j, k, l) and colocalization of Gadd45α immunostaining and positive TUNEL nuclei, Gadd45α/TUNEL (m, n, o). All relevant informations are reported in the figure. Arrowheads indicate TUNEL-positive cells and arrows indicate cell positively immunostained by the Gadd45α mAb. In caspase-3, Gadd45α and Gadd45α/TUNEL panels, original magnification × 400. Human colon NI: uninfected human colon; M90T-infected: EVOC infected over-night with 108 CFU of M90T; BS176-infected: EVOC infected over-night with 108 CFU of BS176

Mentions: To determine the impact of Shigella on epithelial cell survival in the organotypic culture, we analyzed the morphological changes through hematoxylin/eosin (HE) staining. As shown in Figure 6B (panel a), the mucosa architecture of the uninfected samples remained unaltered and only scattered inflammatory cells were interspersed throughout the chorion.


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)

Apoptosis assessment and Gadd45α expression in a human ex vivo organ culture (EVOC) invasion assay with S. flexneri M90T. (A) Dot blot of the distribution of LPS, Gadd45α, TUNEL and caspase-3 immunostained epithelial cells in sections of human colon biopsies infected with M90T or BS176 (12 h) or uninfected. Immunohistochemically stained cells were counted at × 400 magnification. For each sample, five view fields in five sections were considered for cell enumeration. Circles represent the mean values per sample while the horizontal line represents the mean value per sample category. ***P<0.001 after Student's t-test. (B) Histopathological and IH characterization of human colon sections infected with M90T or BS176 or uninfected. Upper panels (HE and LPS): HE (a, b, c) HE staining; LPS (d, e, f): IH staining of S. flexneri 5 LPS, in e and f bacterial LPS are indicated by arrows. In HE original magnification 200; in LPS original magnification × 400. Lower panels: analysis in serial sections of EVOC of immunolabeled mature caspase-3 (g, h, i), Gadd45α (j, k, l) and colocalization of Gadd45α immunostaining and positive TUNEL nuclei, Gadd45α/TUNEL (m, n, o). All relevant informations are reported in the figure. Arrowheads indicate TUNEL-positive cells and arrows indicate cell positively immunostained by the Gadd45α mAb. In caspase-3, Gadd45α and Gadd45α/TUNEL panels, original magnification × 400. Human colon NI: uninfected human colon; M90T-infected: EVOC infected over-night with 108 CFU of M90T; BS176-infected: EVOC infected over-night with 108 CFU of BS176
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Related In: Results  -  Collection

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fig6: Apoptosis assessment and Gadd45α expression in a human ex vivo organ culture (EVOC) invasion assay with S. flexneri M90T. (A) Dot blot of the distribution of LPS, Gadd45α, TUNEL and caspase-3 immunostained epithelial cells in sections of human colon biopsies infected with M90T or BS176 (12 h) or uninfected. Immunohistochemically stained cells were counted at × 400 magnification. For each sample, five view fields in five sections were considered for cell enumeration. Circles represent the mean values per sample while the horizontal line represents the mean value per sample category. ***P<0.001 after Student's t-test. (B) Histopathological and IH characterization of human colon sections infected with M90T or BS176 or uninfected. Upper panels (HE and LPS): HE (a, b, c) HE staining; LPS (d, e, f): IH staining of S. flexneri 5 LPS, in e and f bacterial LPS are indicated by arrows. In HE original magnification 200; in LPS original magnification × 400. Lower panels: analysis in serial sections of EVOC of immunolabeled mature caspase-3 (g, h, i), Gadd45α (j, k, l) and colocalization of Gadd45α immunostaining and positive TUNEL nuclei, Gadd45α/TUNEL (m, n, o). All relevant informations are reported in the figure. Arrowheads indicate TUNEL-positive cells and arrows indicate cell positively immunostained by the Gadd45α mAb. In caspase-3, Gadd45α and Gadd45α/TUNEL panels, original magnification × 400. Human colon NI: uninfected human colon; M90T-infected: EVOC infected over-night with 108 CFU of M90T; BS176-infected: EVOC infected over-night with 108 CFU of BS176
Mentions: To determine the impact of Shigella on epithelial cell survival in the organotypic culture, we analyzed the morphological changes through hematoxylin/eosin (HE) staining. As shown in Figure 6B (panel a), the mucosa architecture of the uninfected samples remained unaltered and only scattered inflammatory cells were interspersed throughout the chorion.

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