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Evolutionary conservation of infection-induced cell death inhibition among Chlamydiales.

Karunakaran K, Mehlitz A, Rudel T - PLoS ONE (2011)

Bottom Line: Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway.Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance.Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Biocenter, University of Würzburg, Würzburg, Germany.

ABSTRACT
Control of host cell death is of paramount importance for the survival and replication of obligate intracellular bacteria. Among these, human pathogenic Chlamydia induces the inhibition of apoptosis in a variety of different host cells by directly interfering with cell death signaling. However, the evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. Here, we investigated the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway. Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.

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Regulation of Bcl-2 family members and PI3 kinase signaling during Simkania infection.(A) Immunoblot analysis of the anti-apoptotic/pro-survival signaling during Simkania infection. HeLa cells were infected with Simkania or Mock (control) in a time course experiment before analysis. Anti-apoptotic Bcl-2 family members like Mcl-1 or Bcl-2 (grey arrowheads) are not regulated. Akt is strongly activated during infection (black arrowheads). Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot analysis of PARP cleavage in Simkania infected cells after treatment with PI3K inhibitor. Simkania infected HeLa 229 cells were treated with Ly294002 (PI3 kinase Inhibitor) on day 3 post infection for 6 hours and induced with 20 ng/ml TNF-α+3 µg/ml Chx or carrier for 4 hours before analysis. PARP cleavage (black arrowheads) was a measure of apoptosis sensitization. Treatment with 0.1 µM LY29004 clearly sensitized infected cells to apoptosis. Hsp-60 and Actin (white arrowhead) were used as loading controls. n = 2. (C) Bar diagram showing samples treated similar as in Figure 5B but prepared for immunofluorescence analysis. Hoechst stained cells were counted to determine the number of apoptotic cells (40× magnification, five fields per sample, n = 2, Error bars = SE). 1 µM LY29004 was found to restore apoptosis sensitivity to ∼70% of control (D) Immunofluorescence of samples treated as described in Figure 5B–C. Samples were stained with Hoechst (blue) and viewed under a fluorescent microscope for counting. Hoechst dye stained both HeLa cell nuclei and Simkania inclusions (yellow mark). White arrowheads mark example apoptotic cells. n = 2.
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pone-0022528-g005: Regulation of Bcl-2 family members and PI3 kinase signaling during Simkania infection.(A) Immunoblot analysis of the anti-apoptotic/pro-survival signaling during Simkania infection. HeLa cells were infected with Simkania or Mock (control) in a time course experiment before analysis. Anti-apoptotic Bcl-2 family members like Mcl-1 or Bcl-2 (grey arrowheads) are not regulated. Akt is strongly activated during infection (black arrowheads). Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot analysis of PARP cleavage in Simkania infected cells after treatment with PI3K inhibitor. Simkania infected HeLa 229 cells were treated with Ly294002 (PI3 kinase Inhibitor) on day 3 post infection for 6 hours and induced with 20 ng/ml TNF-α+3 µg/ml Chx or carrier for 4 hours before analysis. PARP cleavage (black arrowheads) was a measure of apoptosis sensitization. Treatment with 0.1 µM LY29004 clearly sensitized infected cells to apoptosis. Hsp-60 and Actin (white arrowhead) were used as loading controls. n = 2. (C) Bar diagram showing samples treated similar as in Figure 5B but prepared for immunofluorescence analysis. Hoechst stained cells were counted to determine the number of apoptotic cells (40× magnification, five fields per sample, n = 2, Error bars = SE). 1 µM LY29004 was found to restore apoptosis sensitivity to ∼70% of control (D) Immunofluorescence of samples treated as described in Figure 5B–C. Samples were stained with Hoechst (blue) and viewed under a fluorescent microscope for counting. Hoechst dye stained both HeLa cell nuclei and Simkania inclusions (yellow mark). White arrowheads mark example apoptotic cells. n = 2.

Mentions: Resistance of apoptotic signaling upstream of mitochondria has previously been shown to involve Bcl-2 family members in Chlamydia-infected cells. One mechanism involves the degradation of pro-apoptotic BH3-only proteins acting to either directly or indirectly induce Bak and/or Bax oligomerisation and mitochondrial outer membrane permeabilization [46], [52]. We tested whether this is similar in Simkania infection but could not detect any degradation of the BH3-only proteins Bad, Bid, Puma, Bim and Bmf (Figure S4). We also tested for the up-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Mcl-1, as the latter has previously been found to be up-regulated and stabilized in C. trachomatis infection [41], [42]. Interestingly, Mcl-1 and Bcl-2 levels remained constant in a time course infection experiment (Figure 5A), indicating that regulation of these Bcl-2 family members does not account for the apoptosis resistance in Simkania infection.


Evolutionary conservation of infection-induced cell death inhibition among Chlamydiales.

Karunakaran K, Mehlitz A, Rudel T - PLoS ONE (2011)

Regulation of Bcl-2 family members and PI3 kinase signaling during Simkania infection.(A) Immunoblot analysis of the anti-apoptotic/pro-survival signaling during Simkania infection. HeLa cells were infected with Simkania or Mock (control) in a time course experiment before analysis. Anti-apoptotic Bcl-2 family members like Mcl-1 or Bcl-2 (grey arrowheads) are not regulated. Akt is strongly activated during infection (black arrowheads). Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot analysis of PARP cleavage in Simkania infected cells after treatment with PI3K inhibitor. Simkania infected HeLa 229 cells were treated with Ly294002 (PI3 kinase Inhibitor) on day 3 post infection for 6 hours and induced with 20 ng/ml TNF-α+3 µg/ml Chx or carrier for 4 hours before analysis. PARP cleavage (black arrowheads) was a measure of apoptosis sensitization. Treatment with 0.1 µM LY29004 clearly sensitized infected cells to apoptosis. Hsp-60 and Actin (white arrowhead) were used as loading controls. n = 2. (C) Bar diagram showing samples treated similar as in Figure 5B but prepared for immunofluorescence analysis. Hoechst stained cells were counted to determine the number of apoptotic cells (40× magnification, five fields per sample, n = 2, Error bars = SE). 1 µM LY29004 was found to restore apoptosis sensitivity to ∼70% of control (D) Immunofluorescence of samples treated as described in Figure 5B–C. Samples were stained with Hoechst (blue) and viewed under a fluorescent microscope for counting. Hoechst dye stained both HeLa cell nuclei and Simkania inclusions (yellow mark). White arrowheads mark example apoptotic cells. n = 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3142178&req=5

pone-0022528-g005: Regulation of Bcl-2 family members and PI3 kinase signaling during Simkania infection.(A) Immunoblot analysis of the anti-apoptotic/pro-survival signaling during Simkania infection. HeLa cells were infected with Simkania or Mock (control) in a time course experiment before analysis. Anti-apoptotic Bcl-2 family members like Mcl-1 or Bcl-2 (grey arrowheads) are not regulated. Akt is strongly activated during infection (black arrowheads). Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot analysis of PARP cleavage in Simkania infected cells after treatment with PI3K inhibitor. Simkania infected HeLa 229 cells were treated with Ly294002 (PI3 kinase Inhibitor) on day 3 post infection for 6 hours and induced with 20 ng/ml TNF-α+3 µg/ml Chx or carrier for 4 hours before analysis. PARP cleavage (black arrowheads) was a measure of apoptosis sensitization. Treatment with 0.1 µM LY29004 clearly sensitized infected cells to apoptosis. Hsp-60 and Actin (white arrowhead) were used as loading controls. n = 2. (C) Bar diagram showing samples treated similar as in Figure 5B but prepared for immunofluorescence analysis. Hoechst stained cells were counted to determine the number of apoptotic cells (40× magnification, five fields per sample, n = 2, Error bars = SE). 1 µM LY29004 was found to restore apoptosis sensitivity to ∼70% of control (D) Immunofluorescence of samples treated as described in Figure 5B–C. Samples were stained with Hoechst (blue) and viewed under a fluorescent microscope for counting. Hoechst dye stained both HeLa cell nuclei and Simkania inclusions (yellow mark). White arrowheads mark example apoptotic cells. n = 2.
Mentions: Resistance of apoptotic signaling upstream of mitochondria has previously been shown to involve Bcl-2 family members in Chlamydia-infected cells. One mechanism involves the degradation of pro-apoptotic BH3-only proteins acting to either directly or indirectly induce Bak and/or Bax oligomerisation and mitochondrial outer membrane permeabilization [46], [52]. We tested whether this is similar in Simkania infection but could not detect any degradation of the BH3-only proteins Bad, Bid, Puma, Bim and Bmf (Figure S4). We also tested for the up-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Mcl-1, as the latter has previously been found to be up-regulated and stabilized in C. trachomatis infection [41], [42]. Interestingly, Mcl-1 and Bcl-2 levels remained constant in a time course infection experiment (Figure 5A), indicating that regulation of these Bcl-2 family members does not account for the apoptosis resistance in Simkania infection.

Bottom Line: Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway.Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance.Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Biocenter, University of Würzburg, Würzburg, Germany.

ABSTRACT
Control of host cell death is of paramount importance for the survival and replication of obligate intracellular bacteria. Among these, human pathogenic Chlamydia induces the inhibition of apoptosis in a variety of different host cells by directly interfering with cell death signaling. However, the evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. Here, we investigated the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway. Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.

Show MeSH
Related in: MedlinePlus