Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst in Entamoeba invadens

Entamoeba Histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ~20h post induction, and normal nuclear morphology was restored by 72h. Two types of cellular structures were visible by 16h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

Three stages in the development of the cyst wall of the eye pathogen Acanthamoeba castellanii

When deprived of nutrients, trophozoites of the eye pathogen Acanthamoeba castellanii make a cyst wall, which contains cellulose and has two layers connected by cone-shaped ostioles. We recently showed chitin is also present and identified three sets of lectins, which localize to the ectocyst layer (Jonah lectin) or the endocyst layer and ostioles (Luke and Leo lectins). To determine how the cyst wall is made, we examined encysting protists using structured illumination microscopy, probes for glycopolymers, and tags for lectins. In the first stage (3 to 9 hr), cellulose, chitin, and a Jonah lectin were each made in dozens of encystation-specific vesicles. In the second stage (12 to 18 hr), a primordial wall contained both glycopolymers and Jonah lectin, while small, flat ostioles were outlined by a Luke lectin. In the third stage (24 to 36 hr), an ectocyst layer enriched in Jonah lectin was connected to an endocyst layer enriched in Luke and Leo lectins by large, conical ostioles. Jonah and Luke lectins localized to the same places in mature cyst walls (72 hr) independent of the timing of expression. The Jonah lectin and the glycopolymer bound by the lectin were accessible in the ectocyst layer of mature walls. In contrast, Luke and Leo lectins and glycopolymers bound by the lectins were mostly inaccessible in the endocyst layer and ostioles. These results show that cyst wall formation is a tightly choreographed event, in which glycopolymers and lectins combine to form a mature wall with a protected endocyst layer.ImportanceWhile the cyst wall of Acanthamoeba castellanii, cause of eye infections, contains cellulose like plants and chitin like fungi, it is a temporary, protective structure, analogous to spore coats of bacteria. We showed here that, unlike plants and fungi, A. castellanii makes cellulose and chitin in encystation-specific vesicles. The outer and inner layers of cyst walls, which resemble the primary and secondary walls of plant cells, respectively, are connected by unique structures (ostioles) that synchronously develop from small, flat circles to large, conical structures. Cyst wall proteins, which are lectins that bind cellulose and chitin, localize to inner or outer layers independent of the timing of expression. Because of its abundance and accessibility in the outer layer, the Jonah lectin is an excellent target for diagnostic antibodies. A description of the sequence of events during cyst wall development is a starting point for mechanistic studies of its assembly.

Ultrastructural Aspects of Encystation and Cyst-Germination in Phytophthora Parasitica

Encystation in Phytophthora parasitica can be divided into 3 stages. In the first, the zoospores line their peripheries with flattened vesicles and fibrillar vacuoles in preparation for encystation. In the second stage, as the zoospores round up and shed their flagella, an initial wall is produced which takes the form of the mature cyst wall in thickness, but not in density. The participation of the flattened vesicles and fibrillar vacuoles in the formation of this initial wall is suggested by the disappearance of these organelles concomitant with wall formation. The third stage involves the maturation of the cyst wall and occurs only after dictyosomes produce vesicles which move to the cyst periphery and fuse to the plasmalemma. Germ tubes are formed in direct and indirect germination and involve the evagination of the plasmalemma and cyst wall proximal to an accumulation of dictyosome-derived vesicles. These vesicles remain at the germ-tube tip as it extends. In indirect germination the germ tube stops after having attained an average length of 6 µm and the vesicles appear to fuse at the hyphal apex, thus forming a cap. Lomasomes do not appear to be cell organelles with a specific function such as well synthesis, but rather seem to represent aggregations of excess membranous material that have formed as a result of the discharge of vesicles at the cell periphery during wall formation. When dictyosome vesicles are inhibited from forming and moving toward the cell periphery, lomasomes are not formed.

Ultrastructural Changes During Formation and Germination of Microcysts in Polysphondylium Pallidum, a Cellular Slime Mould

Ultrastructural changes during encystation of Polysphondylium pallidum, a cellular slime mould, include an increase in fibrillar material in the cytoplasmic matrix, the formation of cytoplasmic microprojections at the cell periphery and the occurrence of tiny vesicles and some larger vacuoles near the cell periphery. The cyst wall appears first as a fluffy, loose network of fibrils. In the mature cyst it consists of a dense inner and a somewhat looser outer layer. It contains inclusions of apparently cytoplasmic origin. Electron-dense material lines the cell periphery beneath the plasma membrane. Excystment of the myxamoeba is accompanied by a swelling of aggregated vacuoles and polyvesicular bodies, the disappearance of the peripheral cytoplasmic lining, and a general loosening of the wall texture. For a limited period the cisternae of the rough endoplasmic reticulum appear highly dilated. The loosened wall eventually breaks and the myxamoeba emerges by extending pseudopodia through the rupture, leaving the entire cyst wall behind.

THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII (NEFF STRAIN)

Encysting cells of Acanthamoeba castellanii, Neff strain, have been examined with the electron microscope. The wall structure and cytoplasmic changes during encystment are described. The cyst wall is composed of two major layers: a laminar, fibrous exocyst with a variable amount of matrix material, and an endocyst of fine fibrils in a granular matrix. The two layers are normally separated by a space except where they form opercula in the center of ostioles (exits for excysting amebae). An additional amorphous layer is probably present between the wall and the protoplast in the mature cyst. Early in encystment the Golgi complex is enlarged and contains a densely staining material that appears to contribute to wall formation. Vacuoles containing cytoplasmic debris (autolysosomes) are present in encysting cells and the contents of some of the vacuoles are deposited in the developing cyst wall. Lamellate bodies develop in the mitochondria and appear in the cytoplasm. Several changes are associated with the mitochondrial intracristate granule. The nucleus releases small buds into the cytoplasm, and the nucleolus decreases to less than half its original volume. The cytoplasm increases in electron density and its volume is reduced by about 80%. The water expulsion vesicle is the only cellular compartment without dense content in the mature cyst. The volume fractions of lipid droplets, Golgi complex, mitochondria, digestive vacuoles, and autolysosomes have been determined at different stages of encystment by stereological analysis of electron micrographs. By chemical analyses, dry weight, protein, phospholipid, and glycogen are lower and neutral lipid is higher in the mature cyst than in the trophozoite.

DEVELOPMENT AND GERMINATION OF THE AZOTOBACTER CYST

The fine structure of Azotobacter vinelandii has been studied by means of electron microscopy of ultrathin sections made of the encysting and germinating cells. The organisms were fixed with KMnO4 and embedded in epoxy resin. On an encystment medium the rod-shaped bacteria begin to assume an almost spherical form and then bark-like exine appears in 1½ to 2 days. The exine thickens and an electron permeable intine forms between it and the shrinking cell body. In 5 days the intine makes up more than half of the cyst volume and begins to show a definite two-layered structure. Meanwhile the peripheral bodies, which may be extensions of the cell membrane of the vegetative cell, disappear as the encystment progresses. The cell wall and membrane of the vegetative cell remain demonstrable as the confining structure of the shrinking central body of the mature cyst. In this central body lipoidal globules appear together with aggregations of nuclear material. Cyst germination begins with an increase in the size of the central body at the expense of the intine. The nuclear aggregations become more diffuse and the lipoidal globules disappear. The exine may be pushed outward and the bark-like fragments separate as the emerging vegetative cell develops. Invagination of the cell wall and membrane may occur at this stage leading to cell division. Empty exines remain as horseshoe-shaped structures.

Entamoeba aulastomi Nöller

1. Entamoeba aulastomi and Hexamita sp. from Haemopis sanguisuga have been cultivated in inactivated horse-serum diluted 1 : 10 in 0·5 NaCl solution + solid sterile rice-starch, and upon an inspissated horse-serum slope covered with the liquid medium + sterile rice-starch. Cysts of E. aulastomi appeared on or after the 9th day from subcultivation, but cysts of Hexamita have not appeared in the cultures.2. The amoeboid phase of E. aulastomi divides by a simple direct method, without the formation of a spindle. There is no equatorial plate. It was not possible to count the number of chromatin granules.3. The mature cyst is quadrinucleate, though occasional octonucleate cysts are found. Chromatoids are present but are absorbed before hatching. A. large glycogen vacuole is present in the uninucleate cyst. The nucleus divides by the formation of a delicate spindle but no chromosomes were found.4. E. aulastomi are not harmed by being kept at 2° C. but they die out in the 2nd subculture if kept at 30° C. and within 24 hours if kept at 37° C. The cysts also are killed at 37° C.5. If treated with N/20 HCl all amoebae are killed in less than 10 min. but the cysts are unharmed.