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Protochlamydia induces apoptosis of human HEp-2 cells through mitochondrial dysfunction mediated by chlamydial protease-like activity factor.

Matsuo J, Nakamura S, Ito A, Yamazaki T, Ishida K, Hayashi Y, Yoshida M, Takahashi K, Sekizuka T, Takeuchi F, Kuroda M, Nagai H, Hayashida K, Sugimoto C, Yamaguchi H - PLoS ONE (2013)

Bottom Line: A decrease of the mitochondrial membrane potential was also confirmed.Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis.Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Hokkaido, Japan.

ABSTRACT
Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7-1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive chlamydiae.

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HEp-2 cell death induced by Protochlamydia.A) Representative images showing cell death of HEp-2 cells stimulated with bacteria at an MOI of 10, 50 and 100 at 24 h after incubation. Cell death was estimated by morphological nuclear changes as observed by DAPI staining under a fluorescence microscope. Typical morphological changes of segmented nuclei indicate cell death. Negative control; a culture without the bacteria. Staurosporine; a culture with the drug (10 µM). B) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia and dependent on MOI. Cells were cultured with or without the bacteria adjusted to an MOI of 10–100 for up to 24 h. Staurosporine (10 µM); positive control. The number of dead cells was estimated by DAPI staining. Data are the means ± SD from at least three independent experiments performed in triplicate. *p<0.05 vs. without bacteria (NC). C) Representative western blot showing changes of PARP cleavage dependent on MOI. Cells stimulated with the bacteria for 8 h were collected and then subjected to western blotting with an antibody against PARP, an indicator of the apoptosis signaling cascade (See Methods). The presence of cleaved PARP indicates activation of the apoptosis pathway. α-tubulin was used for the internal control. NC; cells incubated without bacteria. Staurosporine (10 µM); positive control. D) Representative western blot showing changes of PARP cleavage dependent on incubation time. Cells stimulated with bacteria were collected at 4, 8 and 12 h after incubation and then subjected to western blotting with an antibody against PARP as mentioned above.
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pone-0056005-g001: HEp-2 cell death induced by Protochlamydia.A) Representative images showing cell death of HEp-2 cells stimulated with bacteria at an MOI of 10, 50 and 100 at 24 h after incubation. Cell death was estimated by morphological nuclear changes as observed by DAPI staining under a fluorescence microscope. Typical morphological changes of segmented nuclei indicate cell death. Negative control; a culture without the bacteria. Staurosporine; a culture with the drug (10 µM). B) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia and dependent on MOI. Cells were cultured with or without the bacteria adjusted to an MOI of 10–100 for up to 24 h. Staurosporine (10 µM); positive control. The number of dead cells was estimated by DAPI staining. Data are the means ± SD from at least three independent experiments performed in triplicate. *p<0.05 vs. without bacteria (NC). C) Representative western blot showing changes of PARP cleavage dependent on MOI. Cells stimulated with the bacteria for 8 h were collected and then subjected to western blotting with an antibody against PARP, an indicator of the apoptosis signaling cascade (See Methods). The presence of cleaved PARP indicates activation of the apoptosis pathway. α-tubulin was used for the internal control. NC; cells incubated without bacteria. Staurosporine (10 µM); positive control. D) Representative western blot showing changes of PARP cleavage dependent on incubation time. Cells stimulated with bacteria were collected at 4, 8 and 12 h after incubation and then subjected to western blotting with an antibody against PARP as mentioned above.

Mentions: We first determined whether apoptosis induction was dependent on bacterial load or timing. As shown in Figure 1A and B, DAPI staining revealed that Protochlamydia obviously induced apoptosis of HEp-2 cells and, as expected, was dependent on bacterial MOI as demonstrated previously [22]. We also confirmed this feature by western blot analysis using PARP cleavage as a marker of apoptosis, which is located downstream of the apoptosis pathway [23], indicating maximum induction of apoptosis at an MOI of 100 (Figure 1C), possibly by the presence of unknown physical limitation on chlamydial adhesion to cells. We next determined the timing of HEp-2 cell apoptosis after incubation with the bacteria. As a result, PARP cleavage began at 8 h after incubation (Figure 1D). Taken together, the data revealed that some effector molecules might be involved in the apoptosis of HEp-2 cells.


Protochlamydia induces apoptosis of human HEp-2 cells through mitochondrial dysfunction mediated by chlamydial protease-like activity factor.

Matsuo J, Nakamura S, Ito A, Yamazaki T, Ishida K, Hayashi Y, Yoshida M, Takahashi K, Sekizuka T, Takeuchi F, Kuroda M, Nagai H, Hayashida K, Sugimoto C, Yamaguchi H - PLoS ONE (2013)

HEp-2 cell death induced by Protochlamydia.A) Representative images showing cell death of HEp-2 cells stimulated with bacteria at an MOI of 10, 50 and 100 at 24 h after incubation. Cell death was estimated by morphological nuclear changes as observed by DAPI staining under a fluorescence microscope. Typical morphological changes of segmented nuclei indicate cell death. Negative control; a culture without the bacteria. Staurosporine; a culture with the drug (10 µM). B) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia and dependent on MOI. Cells were cultured with or without the bacteria adjusted to an MOI of 10–100 for up to 24 h. Staurosporine (10 µM); positive control. The number of dead cells was estimated by DAPI staining. Data are the means ± SD from at least three independent experiments performed in triplicate. *p<0.05 vs. without bacteria (NC). C) Representative western blot showing changes of PARP cleavage dependent on MOI. Cells stimulated with the bacteria for 8 h were collected and then subjected to western blotting with an antibody against PARP, an indicator of the apoptosis signaling cascade (See Methods). The presence of cleaved PARP indicates activation of the apoptosis pathway. α-tubulin was used for the internal control. NC; cells incubated without bacteria. Staurosporine (10 µM); positive control. D) Representative western blot showing changes of PARP cleavage dependent on incubation time. Cells stimulated with bacteria were collected at 4, 8 and 12 h after incubation and then subjected to western blotting with an antibody against PARP as mentioned above.
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Related In: Results  -  Collection

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pone-0056005-g001: HEp-2 cell death induced by Protochlamydia.A) Representative images showing cell death of HEp-2 cells stimulated with bacteria at an MOI of 10, 50 and 100 at 24 h after incubation. Cell death was estimated by morphological nuclear changes as observed by DAPI staining under a fluorescence microscope. Typical morphological changes of segmented nuclei indicate cell death. Negative control; a culture without the bacteria. Staurosporine; a culture with the drug (10 µM). B) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia and dependent on MOI. Cells were cultured with or without the bacteria adjusted to an MOI of 10–100 for up to 24 h. Staurosporine (10 µM); positive control. The number of dead cells was estimated by DAPI staining. Data are the means ± SD from at least three independent experiments performed in triplicate. *p<0.05 vs. without bacteria (NC). C) Representative western blot showing changes of PARP cleavage dependent on MOI. Cells stimulated with the bacteria for 8 h were collected and then subjected to western blotting with an antibody against PARP, an indicator of the apoptosis signaling cascade (See Methods). The presence of cleaved PARP indicates activation of the apoptosis pathway. α-tubulin was used for the internal control. NC; cells incubated without bacteria. Staurosporine (10 µM); positive control. D) Representative western blot showing changes of PARP cleavage dependent on incubation time. Cells stimulated with bacteria were collected at 4, 8 and 12 h after incubation and then subjected to western blotting with an antibody against PARP as mentioned above.
Mentions: We first determined whether apoptosis induction was dependent on bacterial load or timing. As shown in Figure 1A and B, DAPI staining revealed that Protochlamydia obviously induced apoptosis of HEp-2 cells and, as expected, was dependent on bacterial MOI as demonstrated previously [22]. We also confirmed this feature by western blot analysis using PARP cleavage as a marker of apoptosis, which is located downstream of the apoptosis pathway [23], indicating maximum induction of apoptosis at an MOI of 100 (Figure 1C), possibly by the presence of unknown physical limitation on chlamydial adhesion to cells. We next determined the timing of HEp-2 cell apoptosis after incubation with the bacteria. As a result, PARP cleavage began at 8 h after incubation (Figure 1D). Taken together, the data revealed that some effector molecules might be involved in the apoptosis of HEp-2 cells.

Bottom Line: A decrease of the mitochondrial membrane potential was also confirmed.Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis.Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Hokkaido, Japan.

ABSTRACT
Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7-1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive chlamydiae.

Show MeSH
Related in: MedlinePlus