The eukaryotic protozoan Entamoeba gingivalis (E.g.) is strongly associated with inflamed periodontal pockets. Unlike other obligate anaerobic Entamoeba species, it is considered to not have a life cycle of actively dividing trophozoites and dormant cysts. Accordingly, it has been regarded as non-infectious. To investigate if E.g is capable of encystation in response to adverse environmental conditions, we cultivated clinical isolates of E.g. collected from inflamed periodontal pockets in antibiotics for 8 days. The cytomorphological and ultrastructure forms of the amoeba were investigated by transmission and scanning electron microscopy to reveal cyst formation. We observed exocysts and the encapsulated trophozoids separated by an intra-cystic space, a dense poorly vesiculated cytoplasma and polygonal surface areas of cysts. The cysts walls were composed of chitin. Cysts were conspicuously smaller compared to trophozoids and lacked pseudo- and filipodia. We did not observe multi-nucleated trophozoids after antibiotic induces encystation. Cyst formation in E.g may explain why established treatment approaches often do not stop periodontal tissue destruction during periodontitis and periimplantitis.
BackgroundEntamoeba gingivalis (E. gingivalis) is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where it can feed on epithelial cells and elicit a severe innate immune response. Unlike other Entamoeba species, it is considered that E. gingivalis cannot form cysts, because it is a non-infectious protozoan. The lack of encystation capability would make it susceptible to periodontal treatment. However, it is not clear how the human host becomes infected with E. gingivalis trophozoites. We investigated the ability of E. gingivalis to encapsulate in response to an unfavorable environment in vitro.MethodsDifferent strains of E. gingivalis, isolated from inflamed periodontal pocket samples, were cultured for 8 days in the presence or absence of the antimicrobials amoxycillin and metronidazole. To reveal cyst formation, we investigated the morphology and ultrastructure of the amoeba by light, fluorescence, transmission and scanning electron microscopy. We also used the fluorescent dye calcofluor white M2R to demonstrate chitin present in the cyst wall.ResultsWe observed exocysts and an intra-cystic space separating the encapsulated trophozoite from the environment. Remarkably, cysts showed a smooth surface, polygonal edges and smaller size compared to free-living trophozoites. In addition, encapsulated trophozoites that detached from the cyst wall had a dense cytoplasma without phagocytic vesicles. The cyst walls consisted of chitin as in other Entamoba species. The encapsulated trophozoids were mononuclear after antibioticinduced encapsulation.DiscussionWe conclude that E. gingivalis cyst formation has significant implications for dissemination and infection and may explain why established treatment approaches often fail to halt periodontal tissue destruction during periodontitis and peri-implantitis.
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