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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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cIAPs are upregulated on Simkania infection and are required for the resistance of apoptosis.(A) Immunoblot analysis of the anti-apoptotic/pro-survival apoptosis regulators during Simkania infection. HeLa cells were infected with Simkania or Mock (control) (MOI 1) in a time course experiment before analysis. Anti-apoptotic cIAP-1 and -2 (black arrowheads) are strongly upregulated. Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot confirmation of cIAP-1 and -2 knockdown. HeLa cells were transfected with siRNA directed against cIAP-1, -2 or Luciferase (control). cIAP-1 and -2 (black arrowheads) were strongly down regulated at day 3 post transfection. Actin (white arrowheads) was used as loading control. n = 2. (C) Immunoblot analysis of PARP cleavage in Simkania infected cells after cIAP-1 and -2 single or cIAP-1/-2 double knockdown. HeLa 229 cells were transfected for 3 days before Simkania infection. On day 3 post infection cells were 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. Single knockdown of either cIAP-1 or -2 or both clearly sensitized infected cells to apoptosis. Actin (white arrowhead) was used as loading control. n = 2. (D) Bar diagram showing samples treated similar as in Figure 6C 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). Single knockdown of either cIAP-1 or -2 or both was found to restore apoptosis sensitivity to ∼70% of control.
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pone-0022528-g006: cIAPs are upregulated on Simkania infection and are required for the resistance of apoptosis.(A) Immunoblot analysis of the anti-apoptotic/pro-survival apoptosis regulators during Simkania infection. HeLa cells were infected with Simkania or Mock (control) (MOI 1) in a time course experiment before analysis. Anti-apoptotic cIAP-1 and -2 (black arrowheads) are strongly upregulated. Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot confirmation of cIAP-1 and -2 knockdown. HeLa cells were transfected with siRNA directed against cIAP-1, -2 or Luciferase (control). cIAP-1 and -2 (black arrowheads) were strongly down regulated at day 3 post transfection. Actin (white arrowheads) was used as loading control. n = 2. (C) Immunoblot analysis of PARP cleavage in Simkania infected cells after cIAP-1 and -2 single or cIAP-1/-2 double knockdown. HeLa 229 cells were transfected for 3 days before Simkania infection. On day 3 post infection cells were 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. Single knockdown of either cIAP-1 or -2 or both clearly sensitized infected cells to apoptosis. Actin (white arrowhead) was used as loading control. n = 2. (D) Bar diagram showing samples treated similar as in Figure 6C 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). Single knockdown of either cIAP-1 or -2 or both was found to restore apoptosis sensitivity to ∼70% of control.

Mentions: We have previously reported that IAPs play a vital role in apoptosis inhibition of C. pneumoniae- and C. trachomatis-infected cells [43], [44]. Hence, we tested for the regulation of IAPs in Sn infected cells. Interestingly, we found that cIAP-1 and cIAP-2 were up-regulated in the course of infection (Figure 6A). We therefore tested if these IAPs are required to maintain the high apoptosis resistance in infected cells. Expression of cIAP-1 and cIAP-2 was silenced in control and infected cells using specific siRNAs and induced with TNF/Chx for apoptosis. The infected cells were found sensitized for apoptosis in the absence of cIAP-1 and cIAP-2 (Figure 6B–D), supporting their role in blocking apoptosis in infected cells.


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

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

cIAPs are upregulated on Simkania infection and are required for the resistance of apoptosis.(A) Immunoblot analysis of the anti-apoptotic/pro-survival apoptosis regulators during Simkania infection. HeLa cells were infected with Simkania or Mock (control) (MOI 1) in a time course experiment before analysis. Anti-apoptotic cIAP-1 and -2 (black arrowheads) are strongly upregulated. Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot confirmation of cIAP-1 and -2 knockdown. HeLa cells were transfected with siRNA directed against cIAP-1, -2 or Luciferase (control). cIAP-1 and -2 (black arrowheads) were strongly down regulated at day 3 post transfection. Actin (white arrowheads) was used as loading control. n = 2. (C) Immunoblot analysis of PARP cleavage in Simkania infected cells after cIAP-1 and -2 single or cIAP-1/-2 double knockdown. HeLa 229 cells were transfected for 3 days before Simkania infection. On day 3 post infection cells were 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. Single knockdown of either cIAP-1 or -2 or both clearly sensitized infected cells to apoptosis. Actin (white arrowhead) was used as loading control. n = 2. (D) Bar diagram showing samples treated similar as in Figure 6C 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). Single knockdown of either cIAP-1 or -2 or both was found to restore apoptosis sensitivity to ∼70% of control.
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getmorefigures.php?uid=PMC3142178&req=5

pone-0022528-g006: cIAPs are upregulated on Simkania infection and are required for the resistance of apoptosis.(A) Immunoblot analysis of the anti-apoptotic/pro-survival apoptosis regulators during Simkania infection. HeLa cells were infected with Simkania or Mock (control) (MOI 1) in a time course experiment before analysis. Anti-apoptotic cIAP-1 and -2 (black arrowheads) are strongly upregulated. Hsp-60 and Actin (white arrowheads) were used as loading controls. n = 2. (B) Immunoblot confirmation of cIAP-1 and -2 knockdown. HeLa cells were transfected with siRNA directed against cIAP-1, -2 or Luciferase (control). cIAP-1 and -2 (black arrowheads) were strongly down regulated at day 3 post transfection. Actin (white arrowheads) was used as loading control. n = 2. (C) Immunoblot analysis of PARP cleavage in Simkania infected cells after cIAP-1 and -2 single or cIAP-1/-2 double knockdown. HeLa 229 cells were transfected for 3 days before Simkania infection. On day 3 post infection cells were 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. Single knockdown of either cIAP-1 or -2 or both clearly sensitized infected cells to apoptosis. Actin (white arrowhead) was used as loading control. n = 2. (D) Bar diagram showing samples treated similar as in Figure 6C 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). Single knockdown of either cIAP-1 or -2 or both was found to restore apoptosis sensitivity to ∼70% of control.
Mentions: We have previously reported that IAPs play a vital role in apoptosis inhibition of C. pneumoniae- and C. trachomatis-infected cells [43], [44]. Hence, we tested for the regulation of IAPs in Sn infected cells. Interestingly, we found that cIAP-1 and cIAP-2 were up-regulated in the course of infection (Figure 6A). We therefore tested if these IAPs are required to maintain the high apoptosis resistance in infected cells. Expression of cIAP-1 and cIAP-2 was silenced in control and infected cells using specific siRNAs and induced with TNF/Chx for apoptosis. The infected cells were found sensitized for apoptosis in the absence of cIAP-1 and cIAP-2 (Figure 6B–D), supporting their role in blocking apoptosis in infected cells.

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