Cellulose fibrils formation and organisation of cytoskeleton during encystment are essential for Acanthamoeba cyst wall architecture (original) (raw)

Acanthamoebae success as human pathogens is largely due to the highly resistant cysts which represent a crucial problem in treatment of Acanthamoeba infections. Hence, the study of cyst wall composition and encystment play an important role in finding new therapeutic strategies. For the first time, we detected high activity of cytoskeletal elements-microtubular networks and filamentous actin, in late phases of encystment. Cellulose fibrils-the main components of endocyst were demonstrated in inter-cystic space, and finally in the ectocyst, hereby proving the presence of cellulose in both layers of the cyst wall. We detected clustering of intramembranous particles (IMPs) and their density alterations in cytoplasmic membrane during encystment. We propose a hypothesis that in the phase of endocyst formation, the IMP clusters represent cellulose microfibril terminal complexes involved in cellulose synthesis that after cyst wall completion are reduced. Cyst wall impermeability, due largely to a complex polysaccharide (glycans, mainly cellulose) has been shown to be responsible for Acanthamoeba biocide resistance and cellulose biosynthesis pathway is suggested to be a potential target in treatment of Acanthamoeba infections. Disruption of this pathway would affect the synthesis of cyst wall and reduce considerably the resistance to chemotherapeutic agents. Species of free-living amoebae genus Acanthamoeba Volkonsky, 1931 are opportunistic unicellular parasites with worldwide distribution in diverse environments including freshwater, soil, man-made habitats and even clinical settings 1-4. Pathogenic strains are causative agents of usually fatal chronic granulomatous amoebic encephalitis (GAE) and disseminating diseases in immunodeficient individuals and Acanthamoeba keratitis (AK), a painful progressive eye disease in immunocompetent individuals. The constantly rising number of cases of amoebic keratitis is connected with the increasing use of contact lenses and improving awareness 5. To date, not any standard and reliable therapeutic procedures of Acanthamoeba infections have been developed. The treatment of GAE and disseminated infections is limited and only rarely successful 6,7. AK is treated with a series of drugs with various and inconsistent effects, easily manageable treatment is still not available 8,9. The life cycle of Acanthamoeba spp. comprises two stages: an active trophozoite and a dormant, metabolically almost inactive cyst. Trophozoite is a motile stage typical with hyaline spiny subpseudopodia-acanthopodia, produced on the leading pseudopodium and on the entire cell surface 10,11. In unfavourable environmental conditions or in tissues during persistent infections trophozoites encyst 12-14. Cyst stage is typical with a conspicuous double-layered cyst wall, consisting of ectocyst, composed mostly of proteins and polysaccharides, and endocyst, composed mostly of cellulose 15,16. Except for cyst pores (ostioles), these layers are separated by an inter-cystic