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.     

Carbohydrate analysis of Acanthamoeba castellanii

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting “cyst wall” material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.     

EXOCYTOSIS OF LATEX BEADS DURING THE ENCYSTMENT OF ACANTHAMOEBA

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.     

Glucolipid Synthesis in Acanthamoeba castellanii*

SYNOPSIS. Cell-free preparations of Acanthamoeba castellanii trophozoites transfer glucose from UDP-[U-¹⁴C]glucose to a chloroform-soluble form. This radioactive material has been isolated by thin-layer chromatography; it contains an alkali-labile and an alkali-stable (unsaponifiable) component. Treatment of the enzymic product with 0.1 N KOH for 15 min at 0 C or 20 C releases radioactivity into the aqueous phase as glucose. During this treatment, 30–60% of the original glycolipid remains chloroform-soluble. It is considered to be an alkali-stable glycolipid because no further loss of radioactivity occurs during an additional 45-min of treatment with 0.1 N KOH. During incubation with 0.1 N HCI at 100 C glucose is released quantitatively from both the untreated glycolipid and the alkali-stable glycolipid with a half-time of 6 min. Glycolipid formation is inhibited by UDP and is reversible; extracts catalyze the formation of UDP-glucose from the alkali-stable glucolipid and UDP. The chemical and physical properties of the alkali-stable glycolipid are consistent with a glucosyl phosphoryl polyprenol structure. Extracts prepared from cysts catalyze the formation of glycolipids aiso, but the glucosyltransferase activity/cell decreases during the course of encystment. Radioactivity is incorporated into the fraction insoluble in chloroform-methanol-water (1:1:1:) during these incubations when UDP-[U-¹⁴C]glucose or [¹⁴C]glycolipid is the substrate.     

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.     

Short-Cut Pathway to Synthesize Cellulose of Encysting Acanthamoeba

The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.     

Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores

Summary Vigorous agitation caused the zoospores of Phytophthora palmivora to undergo rapid synchronous encystment. The rate of encystment was determined by counting the number of cells with an alkali-resistant cyst wall. 50% of the zoospores formed an alkali-resistant cyst wall within 60 sec of agitation; after 120 sec, essentially all zoospores had encysted. The rate of spontaneous encystment in nonagitated suspensions was much slower. The flagella of nearly all zoospores disappeared within 30 sec of agitation, i.e. prior to the formation of an alkali-resistant cyst wall. Zoospores depend on internal reserves for synthesizing their cyst walls. Approximately 70% of the total carbohydrate in motile zoospores was extracted with water after treating the cells with 70% éthnol. During synchronous encystment, this carbohydrate fraction composed largely of glucans decreased markedly while the insoluble carbohydrate fraction (cyst wall glucan) increased correspondingly. Clearly, the conversion of cytoplasmic glucan into wall glucan plays a major role in zoospore encystment.     

The Physiology of Encystment of Hartmannella castellanii*

SYNOPSIS. Some aspects of the physiology of encystment of the soil amoeba Hartmannella castellanii in a replacement encystment medium consisting of 5 × 10^-2 M MgCl₂ have been investigated. It is suggested that measurement of the cellulose produced during encystment in the synthesis of the cyst wall is a more reliable measure of the process than other methods tried. The degree of encystment was dependent on the physiologic state of the amoebae and the composition of the growth medium, but the initial pH of the encystment medium (C. 4.0-8.5) had little effect on the process. The requirement for Mg during encystment was probably not due to its deficiency during growth. Encystment was inhibited to varying extents by inhibitors of protein synthesis, tetracycline and chloramphenicol and also by arsenate, arsenite and iodoacetate; sodium fluoride, malonate and 2, 4-dinitrophenol were without marked effect. Addition of glucose and α-ketoglutarate to the replacement medium led to improvement in the encystment response. The presence of glutamate and histidine during encystment led to cell death. Other carbon and nitrogen sources had no effect. During encystment there was an increase in the metabolic activity of the amoebae, as measured by their oxygen consumption. This was accompanied by a decrease of about 40% in cellular dry weight and protein content. Of the other chemical components, there were marked initial increases in the levels of total carbohydrates and pentose which were followed by their depletion during cellulose synthesis. Encystment was completed after about 64 hr when the synthesis of cellulose was complete and the oxygen uptake of the amoebae fell to an immeasurable level.     

Studies on the biosynthesis of protein and lipid components of rat liver mitochondria

1. Male rats were injected intraperitoneally with l-[³⁵S]methionine, [³²P]-phosphate and [2-¹⁴C]acetate. The animals were killed at various times up to 72hr. after injection, and liver mitochondria were prepared and fractionated into soluble protein, insoluble protein and lipid for assay of the radioactivity of each fraction. 2. The maximal specific radioactivity of total mitochondrial phospholipid with respect to both ³²P and ¹⁴C was attained after approx. 6hr. 3. ³²P was incorporated most rapidly into phosphatidylethanolamine, maximal incorporation being attained after approx. 6hr.; maximal incorporation into lecithin occurred after 6–12hr. The specific radioactivity of cardiolipin was still slowly increasing at the end of the experiment (72hr.). 4. There were no major differences between the rates of incorporation of ¹⁴C into the lecithin, phosphatidylethanolamine and cardiolipin fractions of mitochondrial phospholipid, maximal incorporation in each case occurring after approx. 6hr. 5. Maximal incorporation of ³⁵S into both soluble and insoluble protein fractions was attained less than 12hr. after injection, the maximal specific radioactivity of soluble protein being higher than that of insoluble protein.     

Correlation of Encystment and Division in Schizopyrenus russelli*

SYNOPSIS. Schizopyrenus russelli, a free-living soil ameba, grows and encysts in the presence of bacteria. The encystment occurs with decline in the division rate. This is accompanied by incorporation of [U-¹⁴C] glucose into cyst cellulose. The degree of multiplication (but not of encystment) is a function of bacterial concentration. Berenil, a trypanocidal drug, while allowing excystment, completely inhibited multiplication of emerged amebae and their encystment. Addition of this drug after 24 hr, when amebae had gone into a phase of active division failed to check encystment, although it still inhibited further multiplication of the amebae. The findings suggest that a phase of cell division may be a prerequisite for encystment.     

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.     

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.     

Biosynthesis of heterocyst glycolipids of Anabaena cylindrica

The incorporation of sodium acetate-[1-¹⁴C] into the heterocyst glycolipids of Anabaena cylindrica cultures from 60–234 hr old is reported. Incorporation of radioactivity was maximal in 88 hr old cultures. In 60 hr and 88 hr cultures about 90 % of the radioactivity of the heterocyst glycolipids was found in the non-saponifiable glycolipid fraction, whereas in older cultures this fraction contained only 75 % of the radioactivity. Acid hydrolysis of non-saponifiable heterocyst glycolipid fractions showed that in 60 hr cultures, 81 % of the radioactivity occurs in the lipid moiety, whereas in older cultures a greater proportion (40–53 %) of the radioactivity was found in the sugar residue. The lipid fraction obtained by acid hydrolysis contained a mixture of labelled long chain mono-, di- and trihydric alcohols. In young (60 hr) cultures the primary alcohol fraction was most heavily labelled (57.3 % of the radioactivity in the non-saponifiable glycosides) with much smaller amounts in the diol and triol (8.4 and 15.1 % respectively), whereas in older cultures (234 hr) the primary alcohol (23.6 %) diol (22.5 %) and triol (18.9 %) fractions contained ca equal amounts of radioactivity.     

Glycogen synthesis in the fungus Neurospora crassa

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [¹⁴C]glucose from ADP-[¹⁴C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose: glycogen glucosyltransferase as compared with those of the already known UDP glucose: glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-[¹⁴C]glucose or ADP-[¹⁴C]glucose released all the radioactivity as maltose after α or β amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[¹⁴C]glucose was the substrate but the stimulation was much greater with ADP-[¹⁴C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent on the presence of a ‘primer’ of the α 1 → 4 glucan type.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Development of glycogen storage ability under cortisol control in primary cultures of rat fetal hepatocytes

Cortisol has been shown to induce glycogen storage function in primary cultures of fetal hepatocytes. The method we describe provides a homogeneous population of hepatocytes by elimination of hematopoietic cells. Hepatocytes transplanted from 15-day-old fetuses were grown in the absence or presence of cortisol (10^?5M) for periods of up to 4 days. In the presence of cortisol, after a lag period (24 hr), the glycogen content increased sharply, regardless of whether the medium was replaced or not. Incorporation of radioactivity from (U) ¹⁴C-glucose into glycogen paralleled glycogen accumulation, but the specific activity of the stored glycogen was lower than the final specific activity of the glucose in the medium. This result shows that free glucose is a good precursor of glycogen but not the only one. Data from chase and labeling experiments prove that the hormone acts on the synthetic pathway. If cortisol was removed the glycogen content dropped, suggesting that glycogen synthesis depends on the continuous presence of the hormone. The in vitro maturation of hepatocyte can be provoked by the hormone before the normal in vivo maturation stage of the onset of glycogen accumulation. Other studies of the same in vivo phenomenon have demonstrated that accumulation of glycogen in the liver prior to birth is corticosteroid dependent, but only an in vitro study could clearly show that the hormone acts at the cellular level.     

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.     

Incorporation of [C]Glucose into Cell Wall Polysaccharides of Cotton Roots: Effects of NaCl and CaCl(2)

Cotton (Gossypium hirsutum L. cv Acala SJ-2) seedlings were grown in nutrient solutions with four combinations of NaCl (0.1 and 150 millimolar) and CaCl₂ (1 and 10 millimolar) for 7 days, and then exposed to [¹⁴C]glucose for 5 hours. Uptake and incorporation of [¹⁴C]glucose into various cell wall fractions of the root tips were determined. At 1 millimolar Ca²⁺, treatment with 150 millimolar NaCl slightly stimulated uptake but considerably inhibited glucose incorporation into noncellulosic and cellulosic polysaccharides. Supplemental Ca²⁺ did not affect incorporation of glucose into the noncellulosic fraction (regardless of NaCl treatment) but completely alleviated the inhibitory effect of NaCl on glucose incorporation into cellulose. We suggest that high Na⁺ concentrations reduce synthesis of cellulose in cotton roots via disturbance of plasma membrane integrity and that supplemental Ca²⁺ counteracts this effect. The effects on cellulose biosynthesis are proposed to be related to Ca²⁺ displacement from the plasma membrane.     

The inhibitory effect of glucose on the differentiation of trophic Hartmannella culbertsoni into viable cysts.

During encystation of Hartmannella culbertsoni induced by taurine or epinephrine, 60-70% of the reserve glycogen is degraded. Glycogen phosphorylase is activated and glycogen synthetase is inhibited after 6-8 hr of exposure to the encystation medium. The carbon skeleton of glycogen but not that of protein is utilised in the synthesis of cyst wall cellulose. Exogenously added glucose (225 and 550 mM) blocks encystation, degradation of glycogen and synthesis of cellulose. Cyclic AMP synthesis is also very much reduced in cells exposed to glucose.