The most abundant cyst wall proteins of Acanthamoeba castellanii are lectins that bind cellulose and localize to distinct structures in developing and mature cyst walls

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.

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

10.1101/534487 ◽

2019 ◽

Author(s):

Pamela Magistrado-Coxen ◽

Yousuf Aqeel ◽

A Lopez ◽

John Samuelson

Keyword(s):

Cyst Wall ◽

Acanthamoeba Castellanii ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Eye Infections ◽

Second Stage ◽

Sequence Of Events ◽

Starting Point ◽

Third Stage ◽

Mature Cyst

AbstractWhen 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.

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Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts

Contact Lens and Anterior Eye ◽

10.1016/j.clae.2015.11.004 ◽

2016 ◽

Vol 39 (3) ◽

pp. 239-243 ◽

Cited By ~ 15

Author(s):

Farhat Abjani ◽

Naveed Ahmed Khan ◽

Farzana Abubakar Yousuf ◽

Ruqaiyyah Siddiqui

Keyword(s):

Contact Lens ◽

Cyst Wall ◽

Acanthamoeba Castellanii ◽

Effective Strategy

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Ultrastructural Aspects of Encystation and Cyst-Germination in Phytophthora Parasitica

Journal of Cell Science ◽

10.1242/jcs.9.1.175 ◽

1971 ◽

Vol 9 (1) ◽

pp. 175-191

Author(s):

D. E. HEMMES ◽

H. R. HOHL

Keyword(s):

Cyst Wall ◽

Germ Tube ◽

Average Length ◽

Cell Periphery ◽

Phytophthora Parasitica ◽

Cell Organelles ◽

Wall Formation ◽

Third Stage ◽

Germ Tubes ◽

Mature Cyst

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.

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The most abundant cyst wall proteins ofAcanthamoeba castellaniiare cellulose-binding lectins from three gene families that localize to distinct structures in cyst walls

10.1101/496307 ◽

2018 ◽

Cited By ~ 1

Author(s):

Pamela Magistrado-Coxen ◽

Yousuf Aqeel ◽

Angelo Lopez ◽

John R. Haserick ◽

Breeanna R. Urbanowicz ◽

...

Keyword(s):

Cyst Wall ◽

Fluorescent Protein ◽

Acanthamoeba Castellanii ◽

Gene Families ◽

Carbohydrate Binding ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Carbohydrate Binding Modules ◽

Green Fluorescent ◽

Cellulose Binding

AbstractAcanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for glycopolymers to show that purified cyst walls ofA. castellaniiretain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight Cys residues each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for glycopolymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and the glycopolymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and their epitopes are mostly inaccessible in the ectocyst layer and ostioles. In summary, the most abundantA. castellaniiCWPs are three sets of lectins, which have conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique carbohydrate-binding modules (8-Cys of Jonah).Author summaryFifty years ago, the cyst wall ofAcanthamoeba castellaniiwas shown to contain cellulose and have an ectocyst layer, an endocyst layer, and conical ostioles that attach them. The goals here were to identify abundant cyst wall proteins (CWPs) and begin to determine how the wall is assembled. We used wheat germ agglutinin to show cyst walls also contain chitin fibrils. When trophozoites are starved of nutrients, they become immotile and make CWPs and glycopolymers in dozens of small vesicles. The primordial cyst wall is composed of a single, thin layer containing cellulose, chitin, and an abundant CWP we called Jonah. The primordial wall also has small, flat ostioles that contain another abundant CWP we called Luke. Jonah (the best candidate for diagnostic antibodies) is accessible in the ectocyst layer of mature cyst walls, while Luke and a third abundant CWP we termed Leo are present but mostly inaccessible in the endocyst layer and ostioles. WhileA. castellaniicyst walls contain cellulose (like plants) and chitin (like fungi), the glycopolymers are made in vesicles rather than at the plasma membrane, and the CWPs (Luke, Leo, and Jonah lectins) are unique to the protist.

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EXOCYTOSIS OF LATEX BEADS DURING THE ENCYSTMENT OF ACANTHAMOEBA

The Journal of Cell Biology ◽

10.1083/jcb.52.1.117 ◽

1972 ◽

Vol 52 (1) ◽

pp. 117-130 ◽

Cited By ~ 13

Author(s):

James R. Stewart ◽

Robert A. Weisman

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Cyst Wall ◽

Actinomycin D ◽

Acanthamoeba Castellanii ◽

Ultrastructural Analysis ◽

Synthetase Activity ◽

Wall Formation ◽

Latex Beads ◽

In Cells

Cells of Acanthamoeba castellanii (Neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. Amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. The encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase activity, the amount of cyst wall polysaccharide formed, or the percentage of cysts formed. Actinomycin D and cycloheximide inhibited encystment as well as bead expulsion. Ultrastructural analysis revealed that the beads, which initially were contained in phagocytic vesicles, were released from the cell by fusion of vesicular membranes with the plasma membrane. Exocytosis was observed in cells after 3 hr of encystment, with most of the beads being lost before cyst wall formation. Each bead-containing vesicle involved in expulsion was conspicuously demarcated by an area of concentrated cytoplasm, which was more homogeneously granular than the surrounding cytoplasm. Beads were not observed in the cytoplasm of mature cysts but were occasionally found in the cyst wall.

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DNA synthesis in growth and encystment of Acanthamoeba castellanii

Journal of Cell Science ◽

10.1242/jcs.17.3.503 ◽

1975 ◽

Vol 17 (3) ◽

pp. 503-515

Author(s):

L.W. Roti ◽

A.R. Stevens

Keyword(s):

Stationary Phase ◽

Dna Synthesis ◽

Cyst Wall ◽

Acanthamoeba Castellanii ◽

The Other ◽

Differentiated Products ◽

Experimental Induction ◽

Reduced Rate ◽

Zero Time ◽

Wall Components

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown. When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on 32PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.

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

Journal of Cell Science ◽

10.1242/jcs.7.1.285 ◽

1970 ◽

Vol 7 (1) ◽

pp. 285-305

Author(s):

H. R. HOHL ◽

L. Y. MIURA-SANTO ◽

D. A. COTTER

Keyword(s):

Cyst Wall ◽

Ultrastructural Changes ◽

Cell Periphery ◽

Cytoplasmic Matrix ◽

Cellular Slime Mould ◽

Slime Mould ◽

Fibrillar Material ◽

Polysphondylium Pallidum ◽

Cellular Slime ◽

Mature Cyst

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.

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