Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Hokkaido, Japan.
PLoS One. 2013;8(2):e56005. doi: 10.1371/journal.pone.0056005. Epub 2013 Feb 11.
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.
专性阿米巴内共生细菌原衣原体（Protochlamydia）具有祖先致病性衣原体的特征，能够在原生动物宿主（如棘阿米巴）中生存，这一进化发生在 0.7 亿至 14 亿年前，但不能在包括人类在内的脊椎动物中生存。这一观察结果提出了一种可能性，即原衣原体与人类细胞的相互作用可能导致一种新的细胞病变效应，从而深入了解宿主-寄生虫关系。此前，我们报道了原衣原体诱导永生化人细胞系 HEp-2 细胞凋亡。在本研究中，我们试图阐明这种凋亡的分子机制。我们首先证实，在受到细菌刺激后，多聚（ADP-核糖）聚合酶（PARP）在 HEp-2 细胞中早期被切割，这一过程依赖于细菌的数量。泛半胱天冬酶抑制剂以及半胱天冬酶-3 和 -9 抑制剂均能抑制 HEp-2 细胞的凋亡。还证实了线粒体膜电位的下降。此外，氯胺酮（一种抑制衣原体蛋白酶样活性因子（CPAF）的抑制剂）阻断了凋亡。细胞松弛素 D 也抑制了凋亡，且该抑制作用依赖于药物浓度，表明细菌进入细胞是诱导凋亡所必需的。有趣的是，耶尔森氏菌 III 型抑制剂（ME0052、ME0053 和 ME0054）对凋亡没有任何影响。我们还证实，本研究中使用的原衣原体具有与 cpaf 基因同源的基因，且沙眼衣原体 CPAF 活性中心的关键残基组氨酸-101 和丝氨酸-499 被保守。因此，我们的结果表明，原衣原体进入细胞后，分泌的 CPAF 诱导线粒体功能障碍，导致膜电位下降，随后 caspase-9、caspase-3 和 PARP 被切割，引发凋亡。更有趣的是，由于沙眼衣原体感染可以阻断凋亡，我们的发现意味着原始衣原体和致病性衣原体之间的 CPAF 具有独特的特征。
