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.     

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.     

Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.     

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

AIMS: This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.     

ATPase-associated oligomycin resistance in Acanthamoeba castellanii

The multiplication rate of "wild-type" (WT) populations of Acanthamoeba castellanii was inhibited 50% by approximately 3 microgram oligomycin/ml; OliR2, an oligomycin resistant cell line, required approximately 27 microgram/ml for the same inhibition. ATPase solubilized from OliR2 mitochondrial fractions required 3--10-fold higher concentrations of oligomycin than did identical WT fractions to achieve 50% inhibition of activity. Resistance was correlated with altered mitochondrial ATPase sensitivity to oligomycin.     

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.     

Changes in transfer ribonucleic acid population of Acanthamoeba castellanii during growth and encystment.

Fifteen aminoacyl-transfer ribonucleic acids (tRNA's) from vegetative cells (trophozoites) and mature cysts of Acanthamoeba castellanii were compared by reversed-phase 5 chromatography. Little or no differences were detected in reversed-phase 5 chromatography elution profiles of alanyl-, arginyl-, isoleucyl-, phenylalanyl-, prolyl-, seryl-, threonyl-, tryptophanyl- and valyl-tRNA's. Significant differences in the relative proportions of isoaccepting species of leucyl-, lysyl-, methionyl-, aspartyl-, histidyl-, and tyrosyl-tRNA's were observed. Based upon the criterion of cyanogen bromide reactivity with the modified nucleoside queuosine, the content of queuosine in aspartyl-tRNA of A, castellanii is significantly greater in mature cysts than in trophozoites. The similarity of change in reversed-phase 5 chromatography elution profiles of aspartyl-, histidyl-, and tyrosyl-tRNA suggests that a common mechanism is responsible for alterations in the chromatographic patterns.     

Apoptosis of Acanthamoeba castellanii Trophozoites Induced by Oleic Acid

Acanthamoeba spp. can be parasitic in certain situations and are responsible for serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis, and cutaneous acanthamoebiasis. We analyzed the fatty acid composition of Acanthamoeba castellanii trophozoites and tested the inhibitory activity of the main fatty acids, oleic acid and arachidonic acid, in vitro. Oleic acid markedly inhibited the growth of A. castellanii, with trophozoite viability of 57.4% at a concentration of 200 μM. Caspase‐3 staining and annexin V assays showed that apoptotic death occurred in A. castellanii trophozoites. Quantitative PCR and dot blot analysis showed increased levels of metacaspase and interleukin‐1β converting enzyme, which is also an indication of apoptosis. In contrast, arachidonic acid showed negligible inhibition of growth of A. castellanii trophozoites. Stimulated expression of Atg3, Atg8 and LC3A/B genes and monodansylcadaverine labeling suggested that oleic acid induces apoptosis by triggering autophagy of trophozoites.     

Encystation in Acanthamoeba castellanii: development of biocide resistance

Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites. Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.     

Chemiluminescence of Acanthamoeba castellanii.

We have investigated ferrocytochrome c-induced proton ejection from reconstituted cytochrome c oxidase-containing vesicles using careful control of the number of enzyme turnovers. Ferrocytochrome c caused the appearance of protons at the vesicle exterior, and this could be abolished by using a protonophore. In addition, its decay was dependent on the permeability of the vesicle membranes to protons and the number of turnovers of the oxidase. These observations indicate that the ejection of protons was the result of genuine translocation. The possibility of this translocation occurring via a Mitchellian loop as a result of the presence of a reduced hydrogen carrier contaminating the enzyme was considered and excluded. Proton-translocating activity in this reconstituted system depended critically on the ratio of enzyme to lipid used in the reconstitution process and we propose a rationale to account for this. We conclude that our data provide strong support for the proposal that cytochrome c oxidase acts as a proton pump and that approx. 0.9 H+ is excluded per ferrocytochrome c molecule oxidized.     

Anaerobiosis-induced differentiation of Acanthamoeba castellanii

Acanthamoeba castellanii is a free living amoeba ubiquitous in soil and also commonly found in aquatic environments. In waterlogged soils, anoxia is quickly established as the dissolved oxygen is consumed by the organisms present. We were interested in the effects of anoxic conditions upon this organism. Batch cultures degassed with N₂ during mid-exponential growth, induced encystation within 12 h of anoxia, and mature cysts were formed within 2–3 days. Excystation (99%) was achieved by subsequent aeration of these cultures after 3–6 days. Anoxia-induced cysts, maintained in anoxic conditions for up to four months, remained viable. Difference spectra, during anaerobiosis, revealed that cytochromes were not lost, suggesting that the organism retains its respiratory components. The growth rate of trophozoites, grown in a chemostat, was dependent on the concentration of O₂ in the head space and glucose uptake increased at lower dissolved O₂ tensions. The results obtained suggest that A. castellanii has a complex adaptive strategy enabling it to cope with microaerobic and anoxic conditions which may be experienced in the environment.     

Response of Acanthamoeba castellanii mitochondria to oxidative stress

The purpose of this study was to examine the effects of oxidative stress caused by hydroperoxide (H(2)O(2)) in the presence of iron ions (Fe(2+)) on mitochondria of the amoeba Acanthamoeba castellanii. We used isolated mitochondria of A. castellanii and exposed them to four levels of H(2)O(2) concentration: 0.5, 5, 15, and 25 mM. We measured basic energetics of mitochondria: oxygen consumption in phosphorylation state (state 3) and resting state (state 4), respiratory coefficient rates (RC), ADP/O ratios, membrane potential (DeltaPsi(m)), ability to accumulate Ca(2+) , and cytochrome c release. Our results show that the increasing concentrations of H(2)O(2) stimulates respiration in states 3 and 4. The highest concentration of H(2)O(2) caused a 3-fold increase in respiration in state 3 compared to the control. Respiratory coefficients and ADP/O ratios decreased with increasing stress conditions. Membrane potential significantly collapsed with increasing hydroperoxide concentration. The ability to accumulate Ca(2+) also decreased with the increasing stress treatment. The lowest stress treatment (0.5 mM H(2)O(2)) significantly decreased oxygen consumption in state 3 and 4, RC, and membrane potential. The ADP/O ratio decreased significantly under 5 mM H(2)O(2) treatment, while Ca(2+) accumulation rate decreased significantly at 15 mM H(2)O(2). We also observed cytochrome c release under increasing stress conditions. However, this release was not linear. These results indicate that as low as 0.5 mM H(2)O(2) with Fe(2+) damage the basic energetics of mitochondria of the unicellular eukaryotic organism Acanthamoeba castellanii.     

Stable transfection of Acanthamoeba castellanii

A simple method for stable transfection of Acanthamoeba castellanii using plasmids which confer resistance to neomycin G418 is described. Expression of neomycin phosphotransferase is driven by the Acanthamoeba TBP gene promoter, and can be monitored by cell growth in the presence of neomycin G418 or by Western blot analysis. Transfected cells can be passaged in the same manner as control cells and can be induced to differentiate into cysts, in which form they maintain resistance to neomycin G418 for at least several weeks, although expression of neomycin phosphotransferase is repressed during encystment. Expression of EGFP or an HA-tagged EGFP-TBP fusion can be driven from the same plasmid, using an additional copy of the Acanthamoeba TBP gene promoter or a deletion mutant. The TBP-EGFP fusion is localized to the nucleus, except in a small proportion of presumptive pre-mitotic cells. EGFP expression can also be driven by the cyst-specific CSP21 gene promoter, which is completely repressed in growing cells but strongly induced in differentiating cells. Transfected cells maintain their phenotype for several weeks, even in the absence of neomycin G418, suggesting that transfected genes are stably integrated within the genome. These results demonstrate the utility of the neomycin resistance based plasmids for stable transfection of Acanthamoeba, and may assist a number of investigations.     

The cytochromes of Acanthamoeba castellanii.

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).