Mr. 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 biocide resistance and cellulose biosynthesis pathway is suggested to be a potential target in treatment of infections. Disruption of this pathway would affect the synthesis of cyst wall and reduce considerably the resistance to chemotherapeutic agents. Introduction Species of free-living amoebae genus Volkonsky, 1931 are opportunistic unicellular parasites with worldwide distribution in diverse environments including freshwater, soil, man-made habitats and even clinical settings1C4. Pathogenic strains are causative agents of usually fatal chronic granulomatous amoebic encephalitis (GAE) and disseminating diseases in immunodeficient individuals and 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 awareness5. To date, not any standard and reliable therapeutic procedures of infections have been developed. The treatment of GAE NCGC00244536 and disseminated infections is limited and only rarely successful6,7. AK is treated with a series of drugs with various and inconsistent effects, easily manageable treatment is still not available8,9. The life cycle of spp. comprises two stages: an active trophozoite and a dormant, metabolically almost inactive cyst. Trophozoite is a motile stage typical with hyaline spiny subpseudopodia C acanthopodia, produced on the leading pseudopodium and on the entire cell surface10,11. In unfavourable environmental conditions or in tissues during persistent infections trophozoites encyst12C14. Cyst stage is typical with a conspicuous double-layered cyst wall, consisting of ectocyst, composed mostly of proteins and polysaccharides, and endocyst, composed mostly RGS14 of cellulose15,16. Except for cyst pores (ostioles), these layers are separated by an inter-cystic space where scattered fibrillar elements are deposited17,18. During the trophozoite C cyst differentiation, besides trophozoite and mature cyst, further two morphological stages were determined by Chvez-Mangua trophozoites the permanent and distinct identification features are lacking, traditional classification of species was based exclusively on the cyst morphology, where three groups were established20. Group I consists of species with large cysts (diameter over 18 m) with smooth or gently wrinkled ectocyst and stellate endocyst widely separated. Group II consists of species with cysts usually with diameter up to 18 m with wrinkled ectocyst and stellate, polygonal, triangular, or oval endocyst, closely apposed or widely separated. This group includes the majority of described species and also the majority of species associated with human infections. Group III consists of species with diameter up to 18 m with thin smooth or slightly wrinkled ectocyst and oval or slightly angular endocyst11. The present classification of the genus is based on 18S rDNA and includes 21 genotypes to date (T1CT21), from which the most frequently associated with human infections is the T4 genotype21C23. Although both the classification approaches are not well coordinated, the morphological classification has proved useful in interpreting molecular clustering of isolates24. At present the data of thorough light microscopic and ultrastructural analyses are absenting in most of the described species and deposited strains, and prevent comprehensive comparison of morphology in particular species. To date, detailed cytomorphological data on cysts or encystment including electron microscopic methods were published only for Neff strain17 and clinical isolate16,19,25, environmental strain of cyst wall using freeze-techniques combined with transmission electron microscopy was performed only in three works focusing on sp28, is responsible for the trophozoite motility, including formation of acanthopodia, phagocytosis and first phases of the cytopathic effect in invaded tissues30,31. Unfortunately, the information on the role of cytoskeletal proteins tubulin, actin and a motor protein myosin is available only for trophozoites32C36 and the situation in cysts remains almost unknown. Inhibitor studies suggested involvement of cytoskeletal rearrangement in encystment37 and three actin-binding proteins were identified during the cyst wall formation implying actin dynamics in the course of encystment38. However, in mature cysts the presence of actin was not established19. A further dimension is that cysts represent a serious problem in NCGC00244536 the treatment of infections as thanks to their wall organisation with a high content of cellulose they NCGC00244536 are highly resistant to various biocides (disinfectants, chemotherapeutic agents)39C41. This leads to complicated restorative procedures also to relapses in a few instances42. Our knowledge of the.
