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Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection.

Kepp O, Gottschalk K, Churin Y, Rajalingam K, Brinkmann V, Machuy N, Kroemer G, Rudel T - PLoS Pathog. (2009)

Bottom Line: Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases.Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively.Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

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Caspase-independent loss of matrix attachment.(A) Phase contrast pictures of one HeLa colony during the time course of an infection with Ngo shows detachment upon 6 h of treatment and apoptotic morphologies upon 9–12 h post-infection. (B,E,F) Caspase activation during infection and effective caspase inhibition by Z-VAD-fmk was shown by immunoblot detection of cleaved caspase-3 and FACS analysis using CaspACE. The FACS data represent the mean±SD of at least three independent experiments. (C) Electron micrographs depicting matrix loss were taken 15 h post-infection. Z-VAD-fmk pretreatment was used to exclude caspases-dependent effects. (D,G) Cytoskeletal changes were visualized by immunofluorescence microscopy using an actin-specific antibody. 3D remodeling of respective confocal z stacks was performed using Imaris software.
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ppat-1000348-g001: Caspase-independent loss of matrix attachment.(A) Phase contrast pictures of one HeLa colony during the time course of an infection with Ngo shows detachment upon 6 h of treatment and apoptotic morphologies upon 9–12 h post-infection. (B,E,F) Caspase activation during infection and effective caspase inhibition by Z-VAD-fmk was shown by immunoblot detection of cleaved caspase-3 and FACS analysis using CaspACE. The FACS data represent the mean±SD of at least three independent experiments. (C) Electron micrographs depicting matrix loss were taken 15 h post-infection. Z-VAD-fmk pretreatment was used to exclude caspases-dependent effects. (D,G) Cytoskeletal changes were visualized by immunofluorescence microscopy using an actin-specific antibody. 3D remodeling of respective confocal z stacks was performed using Imaris software.

Mentions: Exfoliation of epithelial cells has previously been described to be caused by Ngo infection [3],[4],[26]. Since this process resembles anoikis, we further investigated the connection between Ngo-induced cell detachment and apoptosis. HeLa cells were infected with Ngo VPI (N242), a clinical isolate and morphological changes were correlated with the activation of caspases. Detachment from the culture support was visible as soon as 6 to 9 h post-infection (Figure 1A) concomitant with the proteolytic maturation of caspase 3 (Figure 1B). To test whether caspase activity is required for exfoliation, cells were infected in the absence or presence of the pan-caspase inhibitor Z-VAD-fmk and then were analyzed by electron and fluorescence microscopy. In the presence of Z-VAD-fmk, Ngo-infected cells continued to detach yet remained otherwise intact and hence failed to disintegrate by apoptosis (Figure 1C and 1D, Video S1) while the activation of caspases 3 and 7 was blocked during the entire duration of the experiment (Figure 1E and 1F). Detachment was further analyzed by acquiring z stacks of infected cells by laser scanning confocal microscopy and subsequent 3-dimensional remodeling (Figure 1G and Video S2). The detachment and induction of apoptosis is not a general response of these cells to infection stress since the same cell line exhibits marked apoptosis resistance as consequence of infection with C. trachomatis [27],[28]. These results demonstrate that caspases are required for the apoptotic disassembly of Ngo-infected cells but not for their detachment.


Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection.

Kepp O, Gottschalk K, Churin Y, Rajalingam K, Brinkmann V, Machuy N, Kroemer G, Rudel T - PLoS Pathog. (2009)

Caspase-independent loss of matrix attachment.(A) Phase contrast pictures of one HeLa colony during the time course of an infection with Ngo shows detachment upon 6 h of treatment and apoptotic morphologies upon 9–12 h post-infection. (B,E,F) Caspase activation during infection and effective caspase inhibition by Z-VAD-fmk was shown by immunoblot detection of cleaved caspase-3 and FACS analysis using CaspACE. The FACS data represent the mean±SD of at least three independent experiments. (C) Electron micrographs depicting matrix loss were taken 15 h post-infection. Z-VAD-fmk pretreatment was used to exclude caspases-dependent effects. (D,G) Cytoskeletal changes were visualized by immunofluorescence microscopy using an actin-specific antibody. 3D remodeling of respective confocal z stacks was performed using Imaris software.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654407&req=5

ppat-1000348-g001: Caspase-independent loss of matrix attachment.(A) Phase contrast pictures of one HeLa colony during the time course of an infection with Ngo shows detachment upon 6 h of treatment and apoptotic morphologies upon 9–12 h post-infection. (B,E,F) Caspase activation during infection and effective caspase inhibition by Z-VAD-fmk was shown by immunoblot detection of cleaved caspase-3 and FACS analysis using CaspACE. The FACS data represent the mean±SD of at least three independent experiments. (C) Electron micrographs depicting matrix loss were taken 15 h post-infection. Z-VAD-fmk pretreatment was used to exclude caspases-dependent effects. (D,G) Cytoskeletal changes were visualized by immunofluorescence microscopy using an actin-specific antibody. 3D remodeling of respective confocal z stacks was performed using Imaris software.
Mentions: Exfoliation of epithelial cells has previously been described to be caused by Ngo infection [3],[4],[26]. Since this process resembles anoikis, we further investigated the connection between Ngo-induced cell detachment and apoptosis. HeLa cells were infected with Ngo VPI (N242), a clinical isolate and morphological changes were correlated with the activation of caspases. Detachment from the culture support was visible as soon as 6 to 9 h post-infection (Figure 1A) concomitant with the proteolytic maturation of caspase 3 (Figure 1B). To test whether caspase activity is required for exfoliation, cells were infected in the absence or presence of the pan-caspase inhibitor Z-VAD-fmk and then were analyzed by electron and fluorescence microscopy. In the presence of Z-VAD-fmk, Ngo-infected cells continued to detach yet remained otherwise intact and hence failed to disintegrate by apoptosis (Figure 1C and 1D, Video S1) while the activation of caspases 3 and 7 was blocked during the entire duration of the experiment (Figure 1E and 1F). Detachment was further analyzed by acquiring z stacks of infected cells by laser scanning confocal microscopy and subsequent 3-dimensional remodeling (Figure 1G and Video S2). The detachment and induction of apoptosis is not a general response of these cells to infection stress since the same cell line exhibits marked apoptosis resistance as consequence of infection with C. trachomatis [27],[28]. These results demonstrate that caspases are required for the apoptotic disassembly of Ngo-infected cells but not for their detachment.

Bottom Line: Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases.Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively.Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

Show MeSH
Related in: MedlinePlus