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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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Both caspase −3 and −9 inhibitors block apoptosis with mitochondrial dysfunction.A) Effect of caspase inhibitors (caspase −1, −3, −8 and −9) on apoptosis. Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia in presence of each caspase inhibitor. Cells were cultured with bacteria (MOI 100) or staurosporine (10 µM) in the presence or absence of each caspase inhibitors (100 µM) 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. the absence of a caspase inhibitor (DMSO). B) A decrease of mitochondrial membrane integrity was observed in HEp-2 cells incubated with Protochlamydia. The integrity was assessed by a staining method using MitoTracker Red CMXRos (See Methods). Normal mitochondria are strongly stained as red (Negative control) compared with abnormal mitochondria (Staurosporine and Protochlamydia).
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pone-0056005-g003: Both caspase −3 and −9 inhibitors block apoptosis with mitochondrial dysfunction.A) Effect of caspase inhibitors (caspase −1, −3, −8 and −9) on apoptosis. Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia in presence of each caspase inhibitor. Cells were cultured with bacteria (MOI 100) or staurosporine (10 µM) in the presence or absence of each caspase inhibitors (100 µM) 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. the absence of a caspase inhibitor (DMSO). B) A decrease of mitochondrial membrane integrity was observed in HEp-2 cells incubated with Protochlamydia. The integrity was assessed by a staining method using MitoTracker Red CMXRos (See Methods). Normal mitochondria are strongly stained as red (Negative control) compared with abnormal mitochondria (Staurosporine and Protochlamydia).

Mentions: Pathogenic chlamydial CPAF directly contributes to the prevention of apoptosis of infected cells through degradation of BH3-only proteins to maintain infected host cells [19]–[21], which is a possible evolutionary path of pathogenic chlamydiae, revolving mitochondrial dysfunction. We therefore determined whether Protochlamydia could modulate mitochondrial function with activation of caspases and PARP cleavages. Using a DAPI staining assay, we found that a pan-caspase inhibitor obviously blocked Protochlamydia-induced apoptosis, and staurosporine, a stimulator that induces caspase-dependent apoptosis (Figure 2A and B). It was also confirmed by western blotting that the inhibitor blocked PARP cleavage (Figure 2C). Using several specific caspase inhibitors, we next determined which caspase molecule was involved in the induction of apoptosis. The results clearly indicated that caspase-3 and −9 inhibitors, but not caspase-1 and −8 inhibitors, blocked the apoptosis (Figure 3A), suggesting that apoptosis occurred through the mitochondrial pathway and was triggered by mitochondrial dysfunction. We also confirmed a decrease of the mitochondrial membrane potential in HEp-2 cells incubated with Protochlamydia (Figure 3B), suggesting mitochondrial dysfunction. Thus, taken together, we clearly observed that Protochlamydia induces apoptosis by mitochondrial dysfunction followed by activations of caspase-9 and −3, and PARP cleavage.


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)

Both caspase −3 and −9 inhibitors block apoptosis with mitochondrial dysfunction.A) Effect of caspase inhibitors (caspase −1, −3, −8 and −9) on apoptosis. Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia in presence of each caspase inhibitor. Cells were cultured with bacteria (MOI 100) or staurosporine (10 µM) in the presence or absence of each caspase inhibitors (100 µM) 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. the absence of a caspase inhibitor (DMSO). B) A decrease of mitochondrial membrane integrity was observed in HEp-2 cells incubated with Protochlamydia. The integrity was assessed by a staining method using MitoTracker Red CMXRos (See Methods). Normal mitochondria are strongly stained as red (Negative control) compared with abnormal mitochondria (Staurosporine and Protochlamydia).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569409&req=5

pone-0056005-g003: Both caspase −3 and −9 inhibitors block apoptosis with mitochondrial dysfunction.A) Effect of caspase inhibitors (caspase −1, −3, −8 and −9) on apoptosis. Numbers of dead cells in HEp-2 cell cultures induced by the addition of Protochlamydia in presence of each caspase inhibitor. Cells were cultured with bacteria (MOI 100) or staurosporine (10 µM) in the presence or absence of each caspase inhibitors (100 µM) 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. the absence of a caspase inhibitor (DMSO). B) A decrease of mitochondrial membrane integrity was observed in HEp-2 cells incubated with Protochlamydia. The integrity was assessed by a staining method using MitoTracker Red CMXRos (See Methods). Normal mitochondria are strongly stained as red (Negative control) compared with abnormal mitochondria (Staurosporine and Protochlamydia).
Mentions: Pathogenic chlamydial CPAF directly contributes to the prevention of apoptosis of infected cells through degradation of BH3-only proteins to maintain infected host cells [19]–[21], which is a possible evolutionary path of pathogenic chlamydiae, revolving mitochondrial dysfunction. We therefore determined whether Protochlamydia could modulate mitochondrial function with activation of caspases and PARP cleavages. Using a DAPI staining assay, we found that a pan-caspase inhibitor obviously blocked Protochlamydia-induced apoptosis, and staurosporine, a stimulator that induces caspase-dependent apoptosis (Figure 2A and B). It was also confirmed by western blotting that the inhibitor blocked PARP cleavage (Figure 2C). Using several specific caspase inhibitors, we next determined which caspase molecule was involved in the induction of apoptosis. The results clearly indicated that caspase-3 and −9 inhibitors, but not caspase-1 and −8 inhibitors, blocked the apoptosis (Figure 3A), suggesting that apoptosis occurred through the mitochondrial pathway and was triggered by mitochondrial dysfunction. We also confirmed a decrease of the mitochondrial membrane potential in HEp-2 cells incubated with Protochlamydia (Figure 3B), suggesting mitochondrial dysfunction. Thus, taken together, we clearly observed that Protochlamydia induces apoptosis by mitochondrial dysfunction followed by activations of caspase-9 and −3, and PARP cleavage.

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