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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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Bim and Bmf mediate Ngo-induced apoptosis.(A,B) HeLa cells were treated with siRNA, and 72 h post-transfection RNA was prepared and the knockdown efficiency was validated by qRT-PCR. The data represent the mean±SD of three independent experiments. Whole cell lysates were prepared for immunoblot detection of the indicated antigens. (C,D) siRNA-transfected cells were infected with Ngo (N242) for 15 h with an multiplicity of infection (MOI) of 1. The caspase activity was measured by FACS analysis using the CaspACE assay. Data represent the mean±SD of at least three independent experiments. Whole cell lysates were taken at the same time point post-infection and the activity of caspase 3 was analyzed using an anti-cleaved caspase 3 antibody. Equal loading was ensured using the indicated loading control. (E) Puma and Noxa transcriptional regulation was examined by qRT-PCR 15 h post-infection (Ngo) versus non-infected cells (Ctr). Data represent the mean±SD of three independent experiments. (F) Expression of Noxa (siNoxa) and Puma (siPuma) was silenced by RNA interference, and the caspase activity was monitored in infected cells. (G) The cytoskeletal fraction of infected and zVAD pretreated cells were checked together with untreated controls. Bim, Bmf, and the indicated loading control were detected using specific antibodies.
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ppat-1000348-g002: Bim and Bmf mediate Ngo-induced apoptosis.(A,B) HeLa cells were treated with siRNA, and 72 h post-transfection RNA was prepared and the knockdown efficiency was validated by qRT-PCR. The data represent the mean±SD of three independent experiments. Whole cell lysates were prepared for immunoblot detection of the indicated antigens. (C,D) siRNA-transfected cells were infected with Ngo (N242) for 15 h with an multiplicity of infection (MOI) of 1. The caspase activity was measured by FACS analysis using the CaspACE assay. Data represent the mean±SD of at least three independent experiments. Whole cell lysates were taken at the same time point post-infection and the activity of caspase 3 was analyzed using an anti-cleaved caspase 3 antibody. Equal loading was ensured using the indicated loading control. (E) Puma and Noxa transcriptional regulation was examined by qRT-PCR 15 h post-infection (Ngo) versus non-infected cells (Ctr). Data represent the mean±SD of three independent experiments. (F) Expression of Noxa (siNoxa) and Puma (siPuma) was silenced by RNA interference, and the caspase activity was monitored in infected cells. (G) The cytoskeletal fraction of infected and zVAD pretreated cells were checked together with untreated controls. Bim, Bmf, and the indicated loading control were detected using specific antibodies.

Mentions: Since infection of HeLa cells with N242 caused the most prominent effects, we focused on this system to further investigate the mechanisms underlying infection-induced apoptosis. We have previously demonstrated that infection with Neisseria induced the activation of Bak and Bax and finally apoptotic cell death [32]. To delineate the signaling pathway leading to the activation of Bak and Bax, we systematically depleted BH3-only proteins in a RNA interference miniscreen. The knockdown of the siRNAs was validated by quantitative real-time PCR (>75% knockdown at the mRNA level) and immunoblot analysis (>75% knockdown at the protein level) (Figure 2A and 2B). Knockdown of Bim and Bmf (but not that of Bid or Bad) resulted in a significant reduction of effector caspase activity, as measured with a fluorogenic caspase 3/7 substrate or by immunochemical detection of proteolytically mature caspase 3 (Figure 2C and 2D, and Figure S4). Bim and Bmf knockdown specifically inhibited the caspase activation induced by Ngo (Figure 2C and 2D, and Figure S5), yet had no effect on caspase activation induced by the genotoxic agent cisplatin (Figure S5A). Ngo infection failed to induce Puma, Noxa and any of the tested mRNAs for BH3 only proteins (Figure 2E, and Figure S6), although cisplatin was able to activate the transcription of both the Puma and Noxa genes (Figure S5B and Figure S5C). Accordingly, the depletion of Puma or Noxa did not affect caspase-3 activation in Ngo-infected cells (Figure 2F). These data demonstrate that Bim and Bmf are specifically required for the Ngo-triggered activation of caspases.


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)

Bim and Bmf mediate Ngo-induced apoptosis.(A,B) HeLa cells were treated with siRNA, and 72 h post-transfection RNA was prepared and the knockdown efficiency was validated by qRT-PCR. The data represent the mean±SD of three independent experiments. Whole cell lysates were prepared for immunoblot detection of the indicated antigens. (C,D) siRNA-transfected cells were infected with Ngo (N242) for 15 h with an multiplicity of infection (MOI) of 1. The caspase activity was measured by FACS analysis using the CaspACE assay. Data represent the mean±SD of at least three independent experiments. Whole cell lysates were taken at the same time point post-infection and the activity of caspase 3 was analyzed using an anti-cleaved caspase 3 antibody. Equal loading was ensured using the indicated loading control. (E) Puma and Noxa transcriptional regulation was examined by qRT-PCR 15 h post-infection (Ngo) versus non-infected cells (Ctr). Data represent the mean±SD of three independent experiments. (F) Expression of Noxa (siNoxa) and Puma (siPuma) was silenced by RNA interference, and the caspase activity was monitored in infected cells. (G) The cytoskeletal fraction of infected and zVAD pretreated cells were checked together with untreated controls. Bim, Bmf, and the indicated loading control were detected using specific antibodies.
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ppat-1000348-g002: Bim and Bmf mediate Ngo-induced apoptosis.(A,B) HeLa cells were treated with siRNA, and 72 h post-transfection RNA was prepared and the knockdown efficiency was validated by qRT-PCR. The data represent the mean±SD of three independent experiments. Whole cell lysates were prepared for immunoblot detection of the indicated antigens. (C,D) siRNA-transfected cells were infected with Ngo (N242) for 15 h with an multiplicity of infection (MOI) of 1. The caspase activity was measured by FACS analysis using the CaspACE assay. Data represent the mean±SD of at least three independent experiments. Whole cell lysates were taken at the same time point post-infection and the activity of caspase 3 was analyzed using an anti-cleaved caspase 3 antibody. Equal loading was ensured using the indicated loading control. (E) Puma and Noxa transcriptional regulation was examined by qRT-PCR 15 h post-infection (Ngo) versus non-infected cells (Ctr). Data represent the mean±SD of three independent experiments. (F) Expression of Noxa (siNoxa) and Puma (siPuma) was silenced by RNA interference, and the caspase activity was monitored in infected cells. (G) The cytoskeletal fraction of infected and zVAD pretreated cells were checked together with untreated controls. Bim, Bmf, and the indicated loading control were detected using specific antibodies.
Mentions: Since infection of HeLa cells with N242 caused the most prominent effects, we focused on this system to further investigate the mechanisms underlying infection-induced apoptosis. We have previously demonstrated that infection with Neisseria induced the activation of Bak and Bax and finally apoptotic cell death [32]. To delineate the signaling pathway leading to the activation of Bak and Bax, we systematically depleted BH3-only proteins in a RNA interference miniscreen. The knockdown of the siRNAs was validated by quantitative real-time PCR (>75% knockdown at the mRNA level) and immunoblot analysis (>75% knockdown at the protein level) (Figure 2A and 2B). Knockdown of Bim and Bmf (but not that of Bid or Bad) resulted in a significant reduction of effector caspase activity, as measured with a fluorogenic caspase 3/7 substrate or by immunochemical detection of proteolytically mature caspase 3 (Figure 2C and 2D, and Figure S4). Bim and Bmf knockdown specifically inhibited the caspase activation induced by Ngo (Figure 2C and 2D, and Figure S5), yet had no effect on caspase activation induced by the genotoxic agent cisplatin (Figure S5A). Ngo infection failed to induce Puma, Noxa and any of the tested mRNAs for BH3 only proteins (Figure 2E, and Figure S6), although cisplatin was able to activate the transcription of both the Puma and Noxa genes (Figure S5B and Figure S5C). Accordingly, the depletion of Puma or Noxa did not affect caspase-3 activation in Ngo-infected cells (Figure 2F). These data demonstrate that Bim and Bmf are specifically required for the Ngo-triggered activation of caspases.

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