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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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Effect of cytochalasin D on the induction of apoptosis.A) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia (MOI 100) were estimated in the presence or absence of cytochalasin D (0.5–2 µM). Cells were cultured with or without bacteria for up to 24 h. 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. DMSO. B) Representative western blot showing changes of PARP cleavage in the presence of cytochalasin D (0.5–2 µM). DMSO; control. Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP.
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pone-0056005-g004: Effect of cytochalasin D on the induction of apoptosis.A) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia (MOI 100) were estimated in the presence or absence of cytochalasin D (0.5–2 µM). Cells were cultured with or without bacteria for up to 24 h. 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. DMSO. B) Representative western blot showing changes of PARP cleavage in the presence of cytochalasin D (0.5–2 µM). DMSO; control. Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP.

Mentions: We assessed whether the apoptosis induced by Protochlamydia was required for bacterial entry into cells using cytochalasin D, an inhibitor that blocks actin remodeling. As a result, the number of dead cells was significantly decreased by treatment with cytochalasin D, which was dependent on the drug concentration (Figure 4A). We also confirmed that the amount of cleaved PARP was clearly decreased by cytochalasin D treatment (Figure 4B). Taken together, the results indicated that bacterial entry into cells is required to induce apoptosis on HEp-2 cells, suggesting that effector molecules secreted into the cytoplasm by bacteria may be involved in the apoptosis.


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)

Effect of cytochalasin D on the induction of apoptosis.A) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia (MOI 100) were estimated in the presence or absence of cytochalasin D (0.5–2 µM). Cells were cultured with or without bacteria for up to 24 h. 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. DMSO. B) Representative western blot showing changes of PARP cleavage in the presence of cytochalasin D (0.5–2 µM). DMSO; control. Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569409&req=5

pone-0056005-g004: Effect of cytochalasin D on the induction of apoptosis.A) Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia (MOI 100) were estimated in the presence or absence of cytochalasin D (0.5–2 µM). Cells were cultured with or without bacteria for up to 24 h. 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. DMSO. B) Representative western blot showing changes of PARP cleavage in the presence of cytochalasin D (0.5–2 µM). DMSO; control. Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP.
Mentions: We assessed whether the apoptosis induced by Protochlamydia was required for bacterial entry into cells using cytochalasin D, an inhibitor that blocks actin remodeling. As a result, the number of dead cells was significantly decreased by treatment with cytochalasin D, which was dependent on the drug concentration (Figure 4A). We also confirmed that the amount of cleaved PARP was clearly decreased by cytochalasin D treatment (Figure 4B). Taken together, the results indicated that bacterial entry into cells is required to induce apoptosis on HEp-2 cells, suggesting that effector molecules secreted into the cytoplasm by bacteria may be involved in the apoptosis.

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