Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Changes in the Activity of Certain Enzymes of Hartmannella (Culbertson strain A-1) During Encystment*

SYNOPSIS. Hartmannella (Culbertson strain A-1) was found to undergo encystment (80–90% in 72 hr) on a non-nutrient agar containing 0.015 M MgCl₂ and 0.02 M taurine. Encystment was completely inhibited by 1 × 10^?5 M Mitomycin C, or 1 × 10^?7 M cycloheximide or 1 × 10^?6 M Actinomycin D. The ability of the amoebae to consume glucose increased fourfold within 24 hr incubation in this medium. The specific activities of cellulose synthetase, hexosephosphate transaminase and uridine diphosphosphoglucose pyrophosphorylase were also stimulated. Dehydrogenases mediating electron transfer from pyruvate, malate, succinate, α-ketoglutarate and α-glycerophosphate to triphenyltetrazolium and from glucose-6-phosphate to nicotinamide-adenine dinucleotide phosphate were, however, repressed during this period of incubation in the encystment medium. The results suggested that, during encystment of Hartmannella A-1, there was a metabolic switchover and the enzyme machinery of the amoeba was oriented more towards biosynthesis of cyst wall constituents than towards the aerobic breakdown of carbohydrates.     

BIOCHEMICAL CHANGES DURING GROWTH AND ENCYSTMENT OF THE CELLULAR SLIME MOLD POLYSPHONDYLIUM PALLIDUM

The growth of the cellular slime mold, Polysphondylium pallidum, was studied on a semidefined medium in shaken suspension. When the medium contained large quantities of particulate material, growth was more rapid and the cellular size and protein content were smaller than when growth occurred on a medium containing less particulate material. The cellular levels of DNA, RNA, and protein; of lysosomal enzymes (acid phosphatase, acid proteinase); and of peroxisomal enzymes (catalase) were assayed during growth and the subsequent stationary phase that led eventually to encystment. Only DNA remained at a constant cellular level. Encystment of exponentially growing cells could also be initiated by washing them and introducing them into a soluble peptone medium. The rate of encystment was proportional to the osmolarity of this medium. The encystment process was followed with respect to the cellular levels of DNA, RNA, protein, carbohydrates, acid phosphatase, acid β-N-Ac-glucosaminidase, and catalase. The most dramatic change occurred in the cellular cellulose content, which increased by at least an order of magnitude by the time encystment was morphologically complete. It was concluded that the encystment of this slime mold in suspension exhibits a number of biochemical similarities to the development of this and other cellular slime molds on a surface.     

Initiation of Encystment in the Mycetozoan Protostelium mycophaga

SYNOPSIS. Protostelium was cultured monoxenically with the yeast Rhodotorula mucilaginosa on either corn meal agar or in liquid corn meal medium. Nearly synchronous encystment was induced in amoebae by washing them free of yeast and incubating them in an encystment medium devised by Neff et al. (1964) for Acanthamoeba.
The extrinsic requirements for encystment were investigated by replacing various components of the encystment medium with other substances. The results indicate that encystment depends on a high concentration of inorganic monovalent cations and that it occurs best at a basic pH. The effect of the ions does not appear to be strictly osmotic.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

HIGH ENCYSTMENT SUCCESS OF THE DINOFLAGELLATE SCRIPPSIELLA CF. LACHRYMOSA IN CULTURE EXPERIMENTS 1

Close to 100% encystment efficiency and a yield above 10⁵ cysts·mL ^{? 1} were routinely achieved in full strength f/2 medium‐based batch cultures (883 μM NO₃ ^? and 36 μM PO₄ ^{? 3}) of the marine dinoflagellate Scrippsiella cf. lachrymosa Lewis. Increases in cell density led to nutrient depletion in this enriched medium, which was the most likely cause for initiation of cyst formation. Lowering the concentration of either nutrient to 1/10 the initial levels decreased the encystment efficiency, whereas use of ammonium as the N source resulted in both low cell yield and low encystment efficiency. The mandatory dormancy period was ca. 60 days and was not affected by cold dark storage of the cysts. Cysts produced in the initial phase of sexual reproduction were relatively large (length 47 μm, width 31 μm) with a heavy calcareous cover. Cysts produced thereafter lacked apparent calcareous cover and were smaller (length 29 μm, width 19 μm). The decrease of cyst volume (by a factor of 0.24–0.4) suggested strong resource limitation during the course of encystment. However, after the mandatory dormancy period, germination success of the smaller cysts was higher (80%), compared with the larger cysts that had been produced initially (50%). Germling survival (74%) was independent of cyst type but was enhanced by higher nutrient concentration during incubation. The ratio of initial nutrient concentration in the medium to the cyst yield was used as a proxy to estimate the cellular nutrient quota. The conservative estimates of 9 pmol N·cyst ^{? 1} and 0.4 pmol P·cyst ^{? 1} obtained in this manner are at the low end of the range of previous published estimates for other dinoflagellate cysts. Given the high encystment observed in laboratory experiments, we have no reason to assume an inherently lower encystment success in dinoflagellate field populations. Our results do not challenge the low nutrient paradigm for dinoflagellate sexuality. We believe that the high encystment success and cyst yield of this particular species is at least partly due to its ability to achieve very high cell densities in cultures, which evidently leads to nutrient depletion even in f/2 medium.     

The regulation of glycoprotein synthesis by cAMP in Dictyostelium

Dictyostelium discoideum (Dd) 1-H vegetative amoebae exposed to cAMP differentiate into mature stalk cells within 48 h [6]. It was of interest to monitor the patterns of glycoprotein synthesis in the amoebae during the first 5 h of exposure to cAMP and phosphate buffer (PB) controls. Following the exposure period the amoebae were labeled with -[6-³H]fucose. It was determined by both silver grain counts of autoradiographs and scintillation spectroscopy that within minutes cAMP effects an inhibition of [³H]fucose incorporation. However, by 5 h of exposure both experimentals and controls lose a major amount of their labeling capacity based upon the initial PB control value. Vegetative amoebae exposed to cAMP mimics the sparse labeling found in prestalk cells. Prestalk cells synthesize cellulose as a result of cAMP-induced gluconeogenesis and consequently glycoprotein synthesis is reduced. Cellular interactions promoted by cAMP appears to initiate prestalk cell differentiation during the pre-aggregation phase of development. This event is accompanied by a loss in the ability of the aggregating cells to synthesize glycoprotein.     

Correlation of cellulose synthesis in vivo and in vitro during the encystment of Acanthamoeba

Acanthamoeba castellanii differentiates when placed in a starvation medium. The mature cysts formed are characterized by a cellulosic wall synthesized from endogenous sources during encystment. A particulate enzyme system whose specific activity increases some 30-fold during encystment catalyzes the formation of an alkali-soluble and an alkali-insoluble β-(1 → 4)-glucan (cellulose). The activity in vitro of this enzyme extracted from populations of cells during encystment correlates with the formation in vivo of the mature cyst and the alkali-insoluble β-glucan of the cyst wall. The conclusion is based on the following observations:

1.

1. Both alkali-soluble and alkali-insoluble β-glucans similar to the enzymatic products of the isolated β-glucan synthetase occur in cyst walls.     

MACROMOLECULAR SYNTHESES RELATED TO THE REPRODUCTIVE CYST OF TETRAHYMENA PATULA

Macromolecular syntheses in encysted Tetrahymena patula were studied using Feulgen fluorescence cytophotometry, autoradiography, and inhibitors of RNA and protein synthesis. Cycloheximide significantly depressed protein synthesis and D-actinomycin effectively blocked RNA synthesis. Under these conditions, the cells within the cyst were unable to divide. Both cytophotometric measurements and autoradiographic data with tritiated thymidine show that DNA synthesis does not occur during the encystment divisions. Excysted cells placed in nutrient broth medium showed a prolonged generation time after the first cell growth cycle, and by the third generation the mean DNA content per cell was almost triple that of starved excysted cells. These findings indicate that (a) the encystment divisions require RNA and protein synthesis, which are apparently effected through turnover, (b) the encystment division cycles occur in the absence of DNA synthesis, and (c) excysted cells placed in culture medium may go through more than one DNA replication per cell cycle.     

The Characterization of an Adrenergic Signalling System Involved in the Encystment of the Ocular Pathogen Acanthamoeba spp.

The aim of this study was to identify and characterize the receptor system involved in controlling encystment in Acanthamoeba using specific agonists and antagonists and to examine whether endogenous stores of catecholamines are produced by the organism. Acanthamoeba trophozoites suspended in axenic growth medium were exposed to adrenoceptor agonists and antagonists to determine which compounds promoted or prevented encystment. Second, trophozoites were cultured in medium containing a catecholamine synthesis inhibitor to investigate the effect this had on natural encystment. Nonspecific adrenoceptor agonists including epinephrine, isoprotenerol, and the selective β1 adrenoceptor agonist dobutamine were found to cause > 90% encystment of Acanthamoeba trophozoites compared to < 30% with the controls. The selective β1 antagonist metoprolol was able to inhibit epinephrine mediated encystment by > 55%. Cultures of Acanthamoeba with the catecholamine synthesis inhibitor α‐methyl‐p‐tyrosine significantly reduced the level of amoebic encystment compared to controls. In conclusion, Acanthamoeba appear to contain a functional adrenergic receptor system of unknown structure which is involved in initiating the encystment process that can be activated and blocked by β1 agonists and antagonists respectively. Furthermore, the presence of this receptor system in Acanthamoeba indicates that topical β adrenoceptor blockers may be effective adjunct therapy by reducing the transformation of trophozoites into the highly resistant cyst stage.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

Glycogen phosphorylase in Acanthamoeba spp.: determining the role of the enzyme during the encystment process using RNA interference

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 Acanthamoeba encystment may be similar to the previously reported regulation mechanisms in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using RNA interference methods, and the effect of this phenomenon was assessed by light and electron microscopy analyses, calcofluor staining, expression zymogram assays, and Northern and Western blot analyses of both small interfering RNA-treated and control cells. The present report establishes the role of glycogen phosphorylase during the encystment process of Acanthamoeba. Moreover, the obtained results demonstrate that the enzyme is required for cyst wall assembly, mainly for the formation of the cell wall inner layer.     

Encystment-Inducing Factor from Cultures of Acanthamoeba palestinensis

Acanthamoeba palestinensis grown in monolayer cultures encysted almost synchronously at a stationary phase, with a yield of about 80% cysts. Under these growth conditions an encystmentinducing factor was released into the medium by transforming amoebae. Cell-free supernatants induced encystment of amoebae from early-log phase cultures. The not dialyzable encystment factor was resistant to nuclease, protease and trypsin digestion, as well as to boiling, but the activity was almost completely destroyed by autoclaving. Isolation and further characterization of the factor will enable clarification of the mode of its action as a regulator of amoeba-cyst transformation.     

Utilizing the macroporous packed bed for insect cell/baculovirus expression

Due to their high porosity and biocompatibility, polyurethane foam (PUF) and cellulose foam were adopted for insect cell immobilization and baculovirus expression. Spodoptera frugiperda (SF-21) cells were grown within the macroporous matrix and then infected by Autographa californica nuclear polyhedrosis virus (AcNPV) which was encoded with human interleukin-5 (hIL-5) gene. An appropriate initial cell loading density and medium circulation velocity determined from the previous study were applied in this actual cell cultivation experiments to obtain a uniform initial and final axial cell distribution. The growth of insect cells and the expression of baculovirus were successful in the macroporous packed bed systems used. The final average cell density in cellulose foam achieved was 5.2×10⁷?cells/cm³ and 4.3×10⁷?cells/cm³ in PUF. Under the conditions of sufficient nutrition and oxygen supplement, the average productivity of hIL-5 in cellulose foam packed bed bioreactor reached 7.2×10⁷ unit/l-day. With 50% fresh medium replacement after viral infection, the average productivity of hIL-5 in PUF packed bed reached 8.4×10⁷ unit/l-day, about two fold than that without any fresh medium replacement at infection.     

Polyphenol oxidase produced during encystation of Acanthamoeba castellanii

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.     

Balamuthia mandrillaris: staining properties of cysts and trophozoites and the effect of 2,6-dichlorobenzonitrile and calcofluor white on encystment

Here, we determined the staining properties of Balamuthia mandrillaris cysts, and assessed the effect of 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, and calcofluor white, a brightening agent, on its encystment. Periodic acid-Schiff reagent stained the inner wall intensely and middle and outer walls weakly suggesting that the cyst wall of B. mandrillaris may contain glycans. Furthermore, cysts, but not trophozoites, fluoresced when stained with calcofluor white. Calcofluor white and DCB, a cellulose synthesis inhibitor, inhibited B. mandrillaris encystment. This is the first report suggesting possible glycan biosynthesis in B. mandrillaris encystment, and this pathwaymay provide a potentially useful drug target and help improve treatment.     

PHAGOCYTOSIS BY THE CELLULAR SLIME MOLD POLYSPHONDYLIUM PALLIDUM DURING GROWTH AND DEVELOPMENT

The phagocytic ability of amoebae of the cellular slime mold Polysphondylium pallidum, grown in shaken suspension, was examined. An established quantitative assay of the uptake of polystyrene (PS) beads was shown to be valid for this organism. The kinetics of phagocytosis were determined, and estimates of the concentration of PS beads necessary to achieve half-maximal phagocytic velocity (K_p), as well as the maximal velocity itself (V_p^max), were made. Comparison with previously published data on Acanthamoeba and guinea pig leukocytes suggested that the P. pallidum amoebae had the lowest K_p, while the leukocytes had the highest V_p^max. Beads approximately 1 µm in diameter appeared to be the optimal size for ingestion. Simultaneously with phagocytosis, comparable numbers of beads accumulated at the cell surface; this accumulation did not occur when phagocytosis was inhibited. Phagocytosis was depressed by protein in the medium, by increased osmolarity, and by inhibitors of aerobic metabolism. Starvation-initiated development, leading to encystment, was shown to affect the capacity of the cells to phagocytize, mainly by progressively decreasing the time span over which the cells ingested particles at a constant initial rate.     

Synthesis of RNA,Protein, Cellulose,and Mucopolysaccharide and Changes in the Chemical Composition of Hartmannella culbertsoni During Encystment under Axenic Conditions*

Hartmannella culbertsoni trophozoites are transformed into viable cysts on exposure to a non-nutrient agar medium containing 15 mM MgCl₂ and 20 mM taurine. Amebae differentiating in this encystment medium incorporate more uracil-2-¹⁴C into RNA and more leucine-1-¹⁴C or valine-1-¹⁴C into proteins than controls. Encysting organisms incorporate significantly more glucose-U-¹⁴C into cellulose and glucosamine-1-¹⁴C into mucopolysaccharides. Incorporation of glucose-U-¹⁴C into cellulose and of glucosamine-1-¹⁴C into mucopolysaccharides are inhibited by actinomycin D or cycloheximide.     

Polyphenol Oxidase Produced During Encystation of Acanthamoeba castellanii

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a K_m value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation. and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.     

Ammonia and the induction of microcyst differentiation in wild-type and mutant strains of the cellular slime mold Polysphondylium pallidum

Although low levels ($\text{0.16 μ}\text{mole}\text{10}{}^7\text{cells, ml}$) of ammonia are present in vegetative, germinating, and young encystment cultures of wild-type (WS-320) cells, a 12-fold increase in extracellular ammonia occurs during microcyst differentiation in the cellular slime mold Polysphondylium pallidum. When WS-320 amoebae were placed in conditioned medium from other wild-type encystment cultures, microcyst formation was initiated earlier than in control (120 mM KCl) cultures. Isoosmotic solutions containing NH₄Cl and KCl also caused WS-320 cells to encyst earlier. Similar results were obtained with mutant strains. In 120 mM KCl, strains mic-1 and mic-2 produce 21 and 64% microcysts, respectively, while strains PN582 and PN651 do not encyst. The mutant mic-1 secretes wild-type levels of ammonia, while mic-2, PN582, and PN651 all secrete much lower amounts. Conditioned medium and solutions containing NH₄Cl and KCl increased the rate of development and the number of microcysts produced by mic-2 and induced microcyst development in the cystless mutant PN582. Taken together these data indicate that ammonia acts as an inducer of microcyst differentiation.