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.
Highlights
Members of the order Chlamydiales are obligate intracellular bacteria that were discovered about a century ago
Apoptosis of HEp-2 Cells is Dependent on the Multiplicity of Infection (MOI) of Protochlamydia at an Early Stage
And B, DAPI staining revealed that Protochlamydia obviously induced apoptosis of HEp-2 cells and, as expected, was dependent on bacterial MOI as demonstrated previously [22]. We confirmed this feature by western blot analysis using poly (ADP-ribose) polymerase (PARP) cleavage as a marker of apoptosis, which is located downstream of the apoptosis pathway [23], indicating maximum induction of apoptosis at an MOI of 100 (Figure 1C), possibly by the presence of unknown physical limitation on chlamydial adhesion to cells
Summary
Members of the order Chlamydiales are obligate intracellular bacteria that were discovered about a century ago. Ancient chlamydiae diverged into pathogenic and primitive chlamydiae 0.7–1.4 billion years ago, all pathogenic chlamydiae species have co-evolved with their vertebrate hosts and so-called primitive chlamydiae have evolved as endosymbionts of lower eukaryotes, namely free-living amoebae (Acanthamoeba) complete with a powerful bacterial killing mechanism [1,2,3,4]. The genome of representative primitive chlamydia (Protochlamydia UWE25) is not in the process of becoming smaller and has stabilized at 2.4 Mb [4]. This observation implies the possibility that, to overcome stressful conditions, primitive chlamydiae still possess certain molecules that pathogenic chlamydiae have already lost. Comparison of the two chlamydiae, which have evolved separately through different paths and inhabiting niches, is extremely intriguing and may lead to new insights into host-parasite relationships
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