Morphological Changes and the Requirements for Macromolecule Synthesis During Excystment of Acanthamoeba Castellanii (original) (raw)
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Ultrastructural Study of Encystation and Excystation in Acanthamoeba castellanii
Journal of Eukaryotic Microbiology, 2005
Encystation and excystation of Acanthamoeba castellanii were studied by transmission electron microscopy. The differentiation process was induced in asynchronous cultures grown axenically. Cytoplasmic vesicles containing a dense fibrous material very similar in appearance to the cyst wall were observed in trophozoites induced to encyst. When these trophozoites were incubated with calcofluor white m2r, fluorescence was observed in cytoplasmic vesicles, suggesting that the material contained in these vesicles corresponded to cyst wall precursors. Semithin cryosections of mature cysts with the same treatment showed fluorescence in the ectocyst and a less intense fluorescence in the endocyst, suggesting the presence of cellulose in both structures of the cyst wall. In mature cysts induced to excystation, small structures very similar to electron-dense granules (EDG) previously described in other amoebae were frequently observed. The EDGs were either sparsely distributed in the cytoplasm or associated with the cytoplasmic face of the plasma membrane. Many of them were located near the ostiole. In advanced phases of excystation, endocytic activity was suggested by the formation of endocytic structures and the presence of vacuoles with fibrous content similar to that of the cyst wall. Electron-dense granules in the process of dissolution were also observed in these vacuoles. Furthermore, the formation of a pseudopod suggests a displacement of the amoeba toward the ostiole.
Acanthamoeba castellanii cysts: new ultrastructural findings
Parasitology Research, 2013
During Acanthamoeba castellanii trophozoitecysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.
The Journal of Cell Biology, 1976
A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a surcrose-Tris-MgSO4-KC1-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the strat of induced encyst...
Ability of Acanthamoeba Cyst to Excyst at Different Temperatures
Jurnal Sains Kesihatan Malaysia, 2011
This study was carried out to observe thermotolerance ability of Acanthamoeba spp. A total of 32 Acanthamoeba spp. isolates obtained from water taps, sinks, swimming pools and sea water were used. Trophozoites of Acanthamoeba spp. were inoculated onto non-nutrient agar (NNA) seeded with heat-killed Escherichia coli using aseptic technique and incubated for 14 days at 30°C to obtain the cyst. The cysts were subcultured onto new agar plates for thermotolerance test at 37°C and 42°C. The plates were observed until 96 hours after incubation for excystation of Acanthamoeba before being declared negative. Overall, 81.25% of samples were able to excyst at 37°C while 37.5% were able to excyst at 42°C. Thermotolerant Acanthamoeba is associated with high pathogenicity potential.
Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment
Eukaryotic Cell, 2010
Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were p...
Scientific Reports, 2019
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
Acanthamoeba castellanii: Structural basis of the cytopathic mechanisms
Experimental Parasitology, 2006
In this study we report observations on the structural mechanisms of the cytopathic eVect of Acanthamoeba castellanii trophozoites on cultured MDCK cell monolayers. Co-incubations were carried out for a maximum of 24 h. The Wrst evidence of damage to the cell monolayer was detected by measuring the transepithelial resistance of cell monolayers that interacted with the amoebae. At 6 h, transepithelial resistance diminished to 51% and amoebae required 5-6 h to produce evidence of structural injury at the light microscopy level. Following 12 h of incubation, the cell monolayer was severely damaged. After making intimate contact with the surface of target cells, trophozoites detached cells from the substrate, lysed and by means of food-cups ingested the damaged cells. There was no morphological evidence of modiWcations in MDCK cell membranes, membrane fusion or junction formation between the amoeba and host plasma membrane. The lytic capacity of the amoebas appears to be the result of cytotoxic factors secreted by the amoebae since, when monolayers were incubated with conditioned medium, there was also a decrease in the transepithelial resistance. Besides, mechanical injury produced by the attachment and movement of the trophozoites may contribute to the disruption of the cell monolayer. As in other pathogenic amoebae, the cytopathic action of A. castellanii on the cell monolayers can subjectively be separated into four stages: adhesion, cytolysis, phagocytosis, and intracellular degradation.
Eukaryotic Cell, 2008
Acanthamoeba infections are difficult to treat due to often late diagnosis and the lack of effective and specific therapeutic agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage that is highly resistant to the available treatments, causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The latter has been reported to be the major component of the inner cyst wall. It has been demonstrated previously that glycogen is the main source of free glucose for the synthesis of cellulose in Acanthamoeba , partly as glycogen levels fall during the encystment process. In other lower eukaryotes (e.g., Dictyostelium discoideum ), glycogen phosphorylase has been reported to be the main tool used for glycogen breakdown in order to maintain the free glucose levels during the encystment process. Therefore, it was hypothesized that the regulation of the key processes involved in the...
Ultrastructural Study of the Encystation and Excystation Processes in Naegleria sp
Journal of Eukaryotic Microbiology, 2009
Encystation and excystation of Acanthamoeba castellanii were studied by transmission electron microscopy. The differentiation process was induced in asynchronous cultures grown axenically. Cytoplasmic vesicles containing a dense fibrous material very similar in appearance to the cyst wall were observed in trophozoites induced to encyst. When these trophozoites were incubated with calcofluor white m2r, fluorescence was observed in cytoplasmic vesicles, suggesting that the material contained in these vesicles corresponded to cyst wall precursors. Semithin cryosections of mature cysts with the same treatment showed fluorescence in the ectocyst and a less intense fluorescence in the endocyst, suggesting the presence of cellulose in both structures of the cyst wall. In mature cysts induced to excystation, small structures very similar to electron-dense granules (EDG) previously described in other amoebae were frequently observed. The EDGs were either sparsely distributed in the cytoplasm or associated with the cytoplasmic face of the plasma membrane. Many of them were located near the ostiole. In advanced phases of excystation, endocytic activity was suggested by the formation of endocytic structures and the presence of vacuoles with fibrous content similar to that of the cyst wall. Electron-dense granules in the process of dissolution were also observed in these vacuoles. Furthermore, the formation of a pseudopod suggests a displacement of the amoeba toward the ostiole.