Encystment Of Acanthamoeba Castellanii Research Articles

Resistance, biguanide sorption and biguanide-induced pentose leakage during encystment of Acanthamoeba castellanii.

This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. A non-nutrient encystment system was used to investigate the changes in the levels of sorption (uptake) of three non-cysticidal concentrations (10, 20 and 50 microg ml(-1)) of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) as well as their effects on viability and leakage of pentose sugars during the first 36 h of encystment. Trophozoites treated with CHA or PHMB were more sensitive and generally sorbed more of each biocide than cysts. During encystment, the largest increases in resistance developed between 18 and 36 h for both biguanides with the resistance emerging to biguanide concentrations of 10 or 20 microg ml(-1) between 18 and 24 h. At 50 microg ml(-1) resistance emerged between 24 and 36 h. There was a general decrease in biocide sorption during encystment between 0-24 and 0-21 h for CHA and PHMB, respectively, at a concentration of 50 microg ml(-1). The greatest decline in biguanide-induced pentose leakage was between 0 and 12 h. The results suggest that during encystment some of the changes in the susceptibility to CHA or PHMB may be related to decreases in the levels of biocide sorption, which is limited by the developing cyst wall. During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.

Coordinate Regulation of Ribosomal Component Synthesis in Acanthamoeba castellanii: 5S RNA Transcription Is Down Regulated during Encystment by Alteration of TFIIIA Activity

Transcription of large rRNA precursor and 5S RNA were examined during encystment of Acanthamoeba castellanii. Both transcription units are down regulated almost coordinately during this process, though 5S RNA transcription is not as completely shut down as rRNA transcription. The protein components necessary for transcription of 5S RNA and tRNA were determined, and fractions containing transcription factors comparable to TFIIIA, TFIIIB, and TFIIIC, as well as RNA polymerase III and a 3'-end processing activity, were identified. Regulation of 5S RNA transcription could be recapitulated in vitro, and the activities of the required components were compared. In contrast to regulation of precursor rRNA, there is no apparent change during encystment in the activity of the polymerase dedicated to 5S RNA expression. Similarly, the transcriptional and promoter-binding activities of TFIIIC are not altered in parallel with 5S RNA regulation. TFIIIB transcriptional activity is unaltered in encysting cells. In contrast, both the transcriptional and DNA-binding activities of TFIIIA are strongly reduced in nuclear extracts from transcriptionally inactive cells. These results were analyzed in terms of mechanisms for coordinate regulation of rRNA and 5S RNA expression.

Identification of Cyclic AMP-Dependent Protein Kinase Activity in Acanthamoeba

a cellulose layer and forms drug-resistant mature cysts. Acanthamoeba cells increase their production of cyclic AMP (cAMP) in response to activation of the enzyme adenylate cyclase either at the stationary phase of growth or during encystment in nonnutrient medium. This study examines whether cAMP acts through the enzyme cyclic AMP-dependent protein kinase (cAMP-PK) in exerting its regulatory influence during formation of cysts. Growth and encystment of Acanthamoeba castellanii (Neff) are described in a previous publication [1]. Extracts of Acanthamoeba are prepared by breaking cells with glass beads in 10 mM Tris-HCl buffer, pH 7.4; 10 mM magnesium chloride; 1 mM EDTA (TME buffer); and 20 % polyethylene glycol. The pellet obtained after centrifugation at 12,000g for 10 minutes is suspended in TME buffer and centrifuged at 1,500g. The supernatant is used as the source of enzyme. Protein kinase is assayed by measuring the rate of incorporation of phosphate from [32PIATP into Kemptide or protein substrates (histone or protamine) bound to phosphocellulose paper. Kemptide is a synthetic peptide, Leu-Arg-Arg-Ala-ser-Leu-Leu-Gly, and its use eliminates cAMP-independent protein kinase activity from the assay procedures. Acanthamoeba extracts exhibit an in vitro cAMP-PK activity with Kemptide as the acceptor substrate and ATP as the phosphate donor. The cAMP-PK enzyme activity of Acanthamoeba cell supernatant is enhanced fourfold by addition of 10 ,uM cAMP. Figure 1 displays the cAMP dependency of the Kemptide-kinase activity of the supernatant fractions of stationary-phase (96 hours) Acanthamoeba. The half-maximal enzyme activity has been estimated to be at the 20 nM concentration of cAMP. By assaying for enzyme activity in the absence of cAMP and in the presence of saturating amounts of cAMP, one can get the activity ratio. This ratio is an estimate of in vitro enzyme activity (-cAMP activity) and total enzyme activity (+cAMP activity) present in the cell. With Kemptide the activity ratios were ~0.2, whereas with protein substrates the ratios were ~0.8, the higher value being due largely to background activities of other kinases. As shown in table 1, activity ratios of extracts of cells in encystment medium increased from 0.2 to ~0.6 after 12 hours of encystment. This result indicates that the enzyme is fully activated by 12 hours during encystment. Earlier work [2] has established that cellulose synthase activity increased rapidly with extracts prepared from cells after 12-16 hours of encystment. The present study indicates that cAMP-PK is fully activated by 12 hours, a finding indicating that active catalytic subunits

Coordinate regulation of synthesis of ribosomal proteins during encystation of Acanthamoeba castellanii.

The synthesis of ribosomal proteins has been examined during growth and encystment of Acanthamoeba castellanii. Cells have been radiolabelled with [35S]methionine and ribosomal proteins have been extracted either from ribosomes or from total cell extracts. The results show that there is less synthesis of ribosomal proteins relative to total cell proteins soon after transfer of growing cells into non-nutrient medium, suggesting that the synthesis of all ribosomal proteins is under coordinate control. Furthermore, synthesis of ribosomal RNA and ribosomal proteins seems to be regulated coordinately, since within 1 h there is 70-80% decline in synthesis of both. Translation of total RNA in vitro indicates that the relative decrease in the synthesis of ribosomal proteins during development is not only due to a relative decrease in the content of ribosomal protein mRNA. However, a considerable (about 10-fold) and coordinate decrease in the relative content of ribosomal protein mRNA occurs during encystment. These data suggest a difference in the control of the synthesis of total proteins and ribosomal proteins as well as in the regulation of the content of total mRNA and ribosomal protein mRNA. The comparison of patterns of ribosomal proteins indicates that during development no qualitative changes occur. The content of one protein of the small ribosomal subunit is low in growing cells and increases during encystation. Since the differences in the concentration of this protein are neither due to changing levels of the corresponding mRNA nor to changing rates of synthesis, changing affinities of this protein to ribosomes in vivo are suggested.

Adenylate cyclase activity during growth and encystment of Acanthamoeba castellanii

The adenylate cyclase activity of Acanthamoeba castellanii (Neff) was studied in extracts prepared after breaking cells in the Potter-Elvehjem homogenizer. The adenylate cyclase activity of cells is low during the exponential growth phase, but then rises 2–4-fold during the stationary phase to a peak, roughly at the time that cyst forms are detectable in the culture. A 2–4-fold activity rise to a peak also occurs 4–8 h after late log cells are transferred to a non-nutrient encystment medium, a time which is shortly before numbers of cyst forms can be detected in the culture. The pattern of activity observed when stationary phase cells are transferred to encystment medium is complex and depends in part on whether the cultures have exhibited the peak of cyclase activity and have begun to initiate cyst formation prior to the transfer. Within the usual time frame after transfer to encystment medium, early logarithmic phase cells do not exhibit a 2–4-fold rise of cyclase activity and they do not encyst. The results suggest a relationship between encystment and the pattern of rise and fall in cyclase specific activity. Fractionation of the homogenate of trophozoites indicated that adenylate cyclase activity was associated with the particulate microsomal fraction.

Growth- and Differentiation-related Enzyme Changes in Cytoplasmic Membranes of Acanthamoeba castellanii

Enzyme activities of microsomal fractions have been used to examine the behaviour of cytoplasmic membranes during growth deceleration and encystment of Acanthamoeba castellanii. Rough and smooth microsomal protein content increased two- to threefold during the transition from exponential growth phase to stationary phase, but declined again as the organisms encysted. Total activities of rotenone-insensitive NADH- and NADPH-cytochrome c reductases, enzymes associated with endoplasmic reticulum, were two- to fivefold higher in stationary phase organisms than in exponentially growing organisms, but were 10- to 100-fold lower in mature cysts. Specific activities of these enzymes in homogenates and rough and smooth microsomal fractions showed the same pattern of change. Homogenate and microsomal activities of alkaline phosphatase, an enzyme known to be present on the contractile vacuole membrane, were highest in exponentially growing organisms, lower in stationary phase organisms and lower still, by at least eightfold relative to stationary phase cells, in mature cysts. The data indicate that during the transition to stationary phase there is extensive proliferation of cytoplasmic membranes which are subsequently broken down as the cells encyst. The changing activities of the cytochrome c reductases can be attributed to this pattern of membrane synthesis followed by breakdown, although there may also be changes in the abundance of functional enzyme molecules on the membranes. The alterations in alkaline phosphatase activity portray the changing status of the membrane system responsible for water expulsion and reflect a less critical need for osmotic control in the cyst than in the trophozoite.

DNA synthesis in growth and encystment of Acanthamoeba castellanii.

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.