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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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Protochlamydia CPAF is involved in apoptosis induction.A) Effect of lactacystin on apoptosis induced by Protochlamydia. Cells were cultured with bacteria (MOI 100) in the presence or absence lactacystin (2 or 10 µM) for 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 lactacystin. B) Representative western blot showing changes of PARP cleavage induced by Protochlamydia in the presence of lactacystin or Staurosporine as a control (2 or 10 µM). Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP. α-tubulin; internal control.
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pone-0056005-g006: Protochlamydia CPAF is involved in apoptosis induction.A) Effect of lactacystin on apoptosis induced by Protochlamydia. Cells were cultured with bacteria (MOI 100) in the presence or absence lactacystin (2 or 10 µM) for 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 lactacystin. B) Representative western blot showing changes of PARP cleavage induced by Protochlamydia in the presence of lactacystin or Staurosporine as a control (2 or 10 µM). Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP. α-tubulin; internal control.

Mentions: We next assessed whether CPAF is involved in the apoptosis. CPAF was widely conserved among chlamydiae including Protochlamydia R18 used in this study, although similarity scores differed among chlamydiae (Figure 5A). Interestingly, while the similarity scores among pathogenic chlamydiae were very high (% of sequence similarity; 47.4–100), the scores among primitive chlamydiae were surprisingly low (% of sequence similarity; 29–100) (Figure 5A). However, histidine-101 and serine-499 in CPAF (C. trachomatis; DUW_3CX), which are two critical residues in the active center [25], were well conserved between primitive and pathogenic chlamydial CPAFs (Figure 5B). Thus, the data suggested that primitive chlamydial CPAF from Protochlamydia is still active and has a critical role in amoebal and mammalian cells. It is therefore expected that a CPAF inhibitor, lactacystin, which directly binds to and blocks pathological chlamydial CPAF activiy [25], could similarly work for inhibition of primitive chlamydial CPAF activity as well, possibly preventing apoptosis. As a result, lactacystin caused a decrease in the number of dead cells (Figure 6A) and blocked PARP cleavage, which was dependent on drug concentration (Figure 6B), indicating that CPAF was involved in the induction of HEp-2 cell apoptosis. In addition, effect of lactacystin itself on inhibition of apoptosis is minimal because of more like lactacystin acting as an accelerator on apoptosis [26].


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)

Protochlamydia CPAF is involved in apoptosis induction.A) Effect of lactacystin on apoptosis induced by Protochlamydia. Cells were cultured with bacteria (MOI 100) in the presence or absence lactacystin (2 or 10 µM) for 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 lactacystin. B) Representative western blot showing changes of PARP cleavage induced by Protochlamydia in the presence of lactacystin or Staurosporine as a control (2 or 10 µM). Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP. α-tubulin; internal control.
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Related In: Results  -  Collection

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

pone-0056005-g006: Protochlamydia CPAF is involved in apoptosis induction.A) Effect of lactacystin on apoptosis induced by Protochlamydia. Cells were cultured with bacteria (MOI 100) in the presence or absence lactacystin (2 or 10 µM) for 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 lactacystin. B) Representative western blot showing changes of PARP cleavage induced by Protochlamydia in the presence of lactacystin or Staurosporine as a control (2 or 10 µM). Cells stimulated with bacteria were collected at 8 h after incubation and then subjected to western blotting with an antibody against PARP. α-tubulin; internal control.
Mentions: We next assessed whether CPAF is involved in the apoptosis. CPAF was widely conserved among chlamydiae including Protochlamydia R18 used in this study, although similarity scores differed among chlamydiae (Figure 5A). Interestingly, while the similarity scores among pathogenic chlamydiae were very high (% of sequence similarity; 47.4–100), the scores among primitive chlamydiae were surprisingly low (% of sequence similarity; 29–100) (Figure 5A). However, histidine-101 and serine-499 in CPAF (C. trachomatis; DUW_3CX), which are two critical residues in the active center [25], were well conserved between primitive and pathogenic chlamydial CPAFs (Figure 5B). Thus, the data suggested that primitive chlamydial CPAF from Protochlamydia is still active and has a critical role in amoebal and mammalian cells. It is therefore expected that a CPAF inhibitor, lactacystin, which directly binds to and blocks pathological chlamydial CPAF activiy [25], could similarly work for inhibition of primitive chlamydial CPAF activity as well, possibly preventing apoptosis. As a result, lactacystin caused a decrease in the number of dead cells (Figure 6A) and blocked PARP cleavage, which was dependent on drug concentration (Figure 6B), indicating that CPAF was involved in the induction of HEp-2 cell apoptosis. In addition, effect of lactacystin itself on inhibition of apoptosis is minimal because of more like lactacystin acting as an accelerator on apoptosis [26].

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