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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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Comparison of similarity scores and conservation of CPAF active centers among CPAFs.A) Comparison of similarity scores among CPAFs. A high score indicates high similarity (Maximum, 100; Minimum 27). Similarity scores among CPAFs were determined by ClustalW2 (See Methods). *accession numbers. B) Alignment of CPAF amino acid sequences. An alignment of CPAFs was constructed by ClustalW2 (See Methods). *critical amino acids, the histidine-101 and serine-499 of CPAF (C. trachomatis DUW_3CX), which are in the active center for CPAF activity.
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pone-0056005-g005: Comparison of similarity scores and conservation of CPAF active centers among CPAFs.A) Comparison of similarity scores among CPAFs. A high score indicates high similarity (Maximum, 100; Minimum 27). Similarity scores among CPAFs were determined by ClustalW2 (See Methods). *accession numbers. B) Alignment of CPAF amino acid sequences. An alignment of CPAFs was constructed by ClustalW2 (See Methods). *critical amino acids, the histidine-101 and serine-499 of CPAF (C. trachomatis DUW_3CX), which are in the active center for CPAF activity.

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)

Comparison of similarity scores and conservation of CPAF active centers among CPAFs.A) Comparison of similarity scores among CPAFs. A high score indicates high similarity (Maximum, 100; Minimum 27). Similarity scores among CPAFs were determined by ClustalW2 (See Methods). *accession numbers. B) Alignment of CPAF amino acid sequences. An alignment of CPAFs was constructed by ClustalW2 (See Methods). *critical amino acids, the histidine-101 and serine-499 of CPAF (C. trachomatis DUW_3CX), which are in the active center for CPAF activity.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056005-g005: Comparison of similarity scores and conservation of CPAF active centers among CPAFs.A) Comparison of similarity scores among CPAFs. A high score indicates high similarity (Maximum, 100; Minimum 27). Similarity scores among CPAFs were determined by ClustalW2 (See Methods). *accession numbers. B) Alignment of CPAF amino acid sequences. An alignment of CPAFs was constructed by ClustalW2 (See Methods). *critical amino acids, the histidine-101 and serine-499 of CPAF (C. trachomatis DUW_3CX), which are in the active center for CPAF activity.
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