A role for a galactose lectin and its ligands during encystment of Entamoeba

In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgatactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.     

UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 microm). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra. Moreover, regardless of whether the enzyme came from encysting or non-encysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.     

NUCLEAR FEATURES AND EFFECT OF NUTRIENTS ON GYMNODINIUM CATENATUM (DINOPHYCEAE) SEXUAL STAGES1

Gymnodinium catenatum Graham is an unarmored, cyst‐forming dinoflagellate species responsible for outbreaks of paralytic shellfish poisoning. The nuclear development of the cells during the sexual cycle and the effect of different nitrate and phosphate external levels on sexual stages were studied. Nuclear fusion of gametes occurred before or at the same time as cytoplasmic fusion. During this process, either both nuclei migrated to a central area in the sulcal region, or only one of them migrated to the other nucleus. The motile and longitudinally biflagellated zygote presented a large, pear‐shaped nucleus, and either divided or encysted. Planozygotes and germlings underwent similar division processes, which suggested an uncoordinated meiosis in both encysting and non‐encysting zygotes. Encystment in culture was greater under low nitrate and phosphate limitation (L/15) than when only one or neither of these nutrients were added (L‐N, L‐P, and ‐N‐P). However, planozygotes individually monitored achieved the maximum encystment (40%) in a medium with no phosphate or nitrate added (‐N‐P), while most of them divided (70%–90%) in replete (L1) or half‐replete (L‐N and L‐P) media. Low levels of nitrate in the medium of cyst formation promoted a deficient development of the cyst wall. On the other hand, low phosphate levels in the medium of germination prevented both planozygote and germling division and lowered the final germination frequencies of cysts. The minimum dormancy, with an average value of 13.7±5.5 days, was not affected by any of the nutritional conditions studied.     

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

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.     

Acid hydrolase activity during growth and encystment in Acanthamoeba castellanii.

The activity and sedimentation of acid phosphatase (APase), acid deoxyribonuclease (DNase), and acid ribonuclease (RNase) were investigated throughout growth and encystment in Acanthamoeba castellanii. The activities/mg protein of all 3 hydrolases are high in young cultures and decrease to constant levels in postlog cells. The RNase activity/ameba decreases 50% during growth, whereas the activity/cell of both APase and DNase remains constant. The percent sedimentation at 20,000 g of all 3 enzymes gradually increases from about 40% in midlog to a plateau of 80% in postlog cells. During encystment, the sedimentation behavior of RNase differs from that of APase and DNase. Encystment is characterized by a differential decrease in the activity/cell of the 3 hydrolases, with RNase decreasing most rapidly and APase least rapidly. APase is unique in that a transient increase of its specific activity is noted during encystment, even though its activity/cell is decreasing.     

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.     

Azotobacter vinelandii aldehyde dehydrogenase regulated by sigma(54): role in alcohol catabolism and encystment

Encystment in Azotobacter vinelandii is induced by n-butanol or beta-hydroxybutyrate (BHB). We identified a gene, encoding an aldehyde dehydrogenase, that was named aldA. An aldA mutation impaired bacterial growth on n-butanol, ethanol, or hexanol as the sole carbon source. Expression of aldA increased in cells shifted from sucrose to n-butanol and was shown to be dependent on the alternative sigma(54) factor. A mutation in rpoN encoding the sigma(54) factor also impaired growth on alcohols. Encystment on n-butanol, but not on BHB, was impaired in aldA or rpoN mutants, indicating that n-butanol is not an inducer of encystment by itself but must be catabolized in order to induce encystment.     

Acid Hydrolase Activity During Growth and Encystment in Acanthamoeba castellanii*

SYNOPSIS. The activity and sedimentation of acid phosphatase (APase), acid deoxyribonuclease (DNase), and acid ribonuclease (RNase) were investigated throughout growth and encystment in Acanthamoeba castellanii. The activities/mg protein of all 3 hydrolases are high in young cultures and decrease to constant levels in postlog cells. The RNase activity/ ameba decreases 50% during growth, whereas the activity/cell of both APase and DNase remains constant. The percent sedimentation at 20,000 g of all 3 enzymes gradually increases from about 40% in midlog to a plateau of 80% in postlog cells. During encystment, the sedimentation behavior of RNase differs from that of APase and DNase. Encystment is characterized by a differential decrease in the activity/cell of the 3 hydrolases, with RNase decreasing most rapidly and APase least rapidly. APase is unique in that a transient increase of its specific activity is noted during encystment, even though its activity/cell is decreasing.     

Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp

Various Naegleria strains were examined to determine the possible origin and significance of membrane-bound black bodies that were found in all exponentially growing cell populations. The bodies, 40–80 nm in diameter, were distributed randomly in the cytoplasm of Naegleria with ultrastructural features typical of trophozoites. No evidence was obtained that the contents of the black bodies were synthesized in the rough endoplasmic reticulum (ER) and packaged by membranous components, which could be a primitive “Golgi complex” in these amoebae. Examination of cells in various stages of encystment indicated that at least some of the cyst wall material was synthesized and packaged by the rough ER. After condensation into amorphous granules in the cisternae, the cyst wall material appeared in vesicles of the rough ER; these were frequently seen in close proximity to the cell membrane in the vicinity of developing cyst wall. Amorphous granules (～100 nm in diameter), which had variable densities and did not appear to be membrane bound, were seen in the cytoplasm of encysting cells. The substance of these granules also seemed to be incorporated into the cyst wall. The membrane-bound black bodies appeared to be destroyed in lysosomal elements during encystment. The membrane-bound black bodies were concluded to be characteristic of trophozoites and unrelated to encytment of Naegleria.     

Giardia intestinalis: Expression of ubiquitin, glucosamine-6-phosphate and cyst wall protein genes during the encystment process

We determined the relative expression of ubiquitin (ub), glucosamine-6-phosphate-isomerase (gn6pi) and cyst wall protein (cwp) genes during encystment of the Portland-1 and Portland-1R strains of Giardia intestinalis. Encystment was induced with bile for different time periods. The presence of encystment-specific vesicles (ESVs) and the relative expression of genes (log₁₀ΔRn) were determined by transmission electron microscopy and real-time PCR, respectively. Our results demonstrated the gene expression and the presence of ESVs after 6 h of encystment. Values of cwp2 gene expression increased by 591-fold in strain Portland-1 and 78.2-fold in strain Portland-1R at this time point compared to values at 0 h, after which values gradually decreased until reaching basal values between 8 and 18 h after the encystment started. Expression of gn6pi was 43.5- and 46.3-fold higher than basal values, in Portland-1 and Portland-1R, respectively. Ub gene expression was 82.25-fold higher than its basal levels at 4 h, after which expression decreased gradually until reaching basal values after 16 h. Conclusions: This work showed the relationship between the presence of ESVs and encystment gene expression at 6 h, and resistance to albendazole does not inhibit the encystment process. The results revealed important knowledge with implications in the control of parasite dissemination for preventing parasite transmission.     

Intracellular polyamine patterns during encystment of Physarum flavicomum

In Physarum flavicomum Berk., growing amoebae convert to dormant cysts under conditions of nutrient imbalance. Exogenous adenine inhibits the process and the cells produce an elevated intracellular concentration of S-adenosylmethionine. Evidence indicates that the increased level of S-adenosylmethionine is responsible for the disruption of the normal developmental process. One of the biological functions of S-adenosylmethionine is in polyamine synthesis and it is known that a well-controlled intracellular concentration of polyamines is essential for normal cell growth and differentiation. In this study, high-performance liquid chromatography was used to determine the intracellular polyamine patterns in growing cells, adenine-treated and normal encysting cells, and dormant cysts. Putrescine and spermidine were the most abundant polyamines found in the cells; growing cells had the highest level, adenine-treated cells had a 1.5 to 2.0 times higher level than normal encysting cells, while cysts had the lowest (only 3 and 12% of that of growing cells). Cadaverine and N1-acetylspermidine were found in all the cells and their levels decreased during encystment. Acetylputrescine was found in growing cells only and acetylspermine was found in all cells except cysts. Acetylcadaverine, N8-acetylspermidine, 1,3-diaminopropane, and spermine were not detected in any of the cells.     

SEXUALITY AND CYST FORMATION IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS: CYST YIELD IN BATCH CULTURES1

Encystment of the toxic dinoflagellate Gonyaulax tamarensis Lebour (var. excavata) was monitored in batch cultures exposed to a variety of nutritional and environmental treatments. Limitation by nitrogen (as ammonium or nitrate) or phosphorus (as phosphate) resulted in cyst formation. When the initial concentration of limiting nutrient was varied, total cyst yield (mL^?1) was directly proportional to the cell yield at all but the highest nutrient concentrations (where encystment was minimal). Encystment efficiency was relatively constant (0.1–0.2 cysts · cell^?1) over a 5-fold range of cell densities, indicating that 20 to 40% of the vegetative populations successfully encysted. Cyst formation was negligible in nutrient-replete medium, even with a significant reduction in growth rate due to non-optimal light, temperature, or to high batch culture cell densities. Low light levels did decrease cyst yield once encystment was initiated by nutrient limitation, but this was probably linked to smaller motile cell yield and not to a specific inhibition of encystment. In contrast, encystment was more sensitive to temperature than was growth rate: optimal cyst production occurred over a relatively narrow temperature range and no cysts were formed at [Page missing]     

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.     

Cyst production and brown pigment formation in aging cultures of Azospirillum brasilense ATCC 29145.

Encystation in Azospirillum brasilense ATCC 29145 was observed by using routine laboratory staining and phase-contrast and electron microscopy. Encystment occurred in liquid and in solid or semisolid media containing fructose (8 mM) and KNO3 (0.5 mM). The encysted forms consisted of a central body filled with poly-beta-hydroxybutyric acid granules, an electron-transparent intinelike region, and a thick outer layer. Enlarged giant encysted forms with multiple central bodies were also observed during the germination of a desiccated brown colony. Morphogenetically different forms in an aging culture could be resolved by sucrose density gradient centrifugation. The dense encysted forms along with numerous granules in a fibrillar network pelleted at 70% sucrose, while empty saclike envelopes along with vegetative cells and coccoid bodies pelleted at 55% sucrose. Different media induced various degrees of pigmentation in A. brasilense ATCC 29145 after aging. The pigment possessed several of the properties reported for microbial melanins, including insolubility in water and organic solvents, solubility in cold and hot alkali, and bleaching in hydrogen peroxide. The UV absorption maxima of the alkali extract were at 280 and 310 nm. Electron micrographs of the brown pigment showed that it occurred as aggregated granules surrounding the encysting cells as well as being excreted into the medium in an aging culture. It is concluded that A. brasilense ATCC 29145 produces compounds that form a brown pigment similar to melanin and are expressed under the influence of certain cultural conditions conducive for encystment.     

Regulation of carbohydrate metabolism during Giardia encystment

Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi1 is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by "cyst wall synthase" (beta 1 --> 3 GalNAc transferase) into a highly insoluble beta 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.     

Induction of Synchronized Fissions in Telotrochidium henneguyi

SYNOPSIS. Cultures of Telotrochidium henneguyi , begun with logarithmic phase cells, were employed in an effort to produce synchronized fission by heat treatment. The cells tolerated a temperature range of 20–50 C; temperatures above 50 were lethal. When cells were exposed to a single shock for 30 min, 30–40 produced 0–50% encystment with total excystment after 10 min exposure to room temperature (heat shock range). No encystment occurred between 20–30 (intershock range). Encystment and excystment time varied directly with temperature between 40–50.
The most effective procedure for inducing synchronized fission consisted of 6 cycle program of 38/28 C (shock temperature/intershock temperature) administered for 15/15 (shock/intershock duration in min). Division indices (DI = cells dividing/total population X 100 =%) ranged from 12–66% with a mean of 37.25%. In control cells, division indices ranged from 2–20% with an average of 12%. Inferences from these independently derived findings are discussed.     

Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation

To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.     

Potential involvement of extracellular signal-regulated kinase 1 and 2 in encystation of a primitive eukaryote,Giardia lamblia. Stage-specific activation and intracellular localization

Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44- and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is approximately 5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial approximately 2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.     

Defective zoospore encystment and suppressed cyst germination of Phytophthora palmivora caused by transient leaching treatments

The behaviour of encysting zoospores of Phytophthora palmivora during leaching conditions was studied. Zoospores encysted and germinated successfully on polycarbonate membranes after mechanical agitation. Transient (10 min) leaching treatments with nutrient-free buffer underneath the membranes resulted in abnormal encystment and poor germination. The disruption was greatest when leaching was applied during the first minutes after start of encystment and not observed after 20 min. The early sensitivity of cells to leaching coincided with the period when alkali-resistant cell walls were formed (2 – 6 min after mechanical agitation). Effects of calcium and organic nutrients on encystment during leaching and germination after these treatments were studied. The disruption of encystment by early leaching treatments, but not the suppression of cyst germination, was overcome by adding calcium chloride during mechanical agitation of zoospores. Leaching with calcium containing buffer resulted in suppressed cyst germination as was the case with buffer alone. Leaching with 0.1 % peptone containing buffer promoted consistently high encystment and germination. This revised version was published online in June 2006 with corrections to the Cover Date.