Acanthamoeba Castellanii Trophozoites Research Articles

Purine salvage by Acanthamoeba castellanii

Trophozoites of Acanthamoeba castellanii were found to incorporate a range of purine bases and nucleosides into parasite nucleic acids. Results from competition studies suggest that A. castellanii is capable of interconverting purine nucleotides. The amoebae contain deaminase, phosphorylase, kinase, phosphoribosyltransferase and 5'-nucleotidase activities towards a number of purine compounds. The results of both the incorporation studies and the enzyme analyses suggest that hypoxanthine is of central importance in the parasite's purine metabolism.

Necessity of iron for the alternative respiratory pathway in acanthamoeba castellanii

Ommission of iron from the growth medium leads, within two to three generations, to a shift in respiration of trophozoites of Acanthamoeba castellanii from the cyanide-stimulated to the cyanide-inhibited pattern. Addition of iron restores the activity of the alternative respiratory pathway within one hour.

DNA-dependent RNA polymerase II from Acanthamoeba castellanii Comparison of the catalytic properties and subunit architectures of the trophozoite and cyst enzymes

The actively growing cells (trophozoites) of the amoeba Acanthamoeba castellanii were found to contain three or perhaps four different forms of class II DNA-dependent RNA polymerase (EC 2.7.7.6). The chromatographic and catalytic properties of all forms of the Acanthamoeba class II polymerases suggest them to be cognates of the class II polymerases previously reported. The predominant form was purified to near homogeneity and its subunit composition determined. Nine different polypeptides were found associated with the purified enzyme: 210 000; 185 000; 140 000; 70 000; 35 000; 21 000; 19 000; 18 500 and 16 200. These polypeptides were interpreted in terms of two class II RNA polymerases which differ in the molecular weight of their largest subunit. When A. castellanii is transferred to a medium lacking nutrients, the cells undergo cellular differentiation resulting in the formation of metabolically inactive cells (cyst formation). During this process there are significant changes in the RNA sequences transcribed. In contrast to this, we find that the chromatographic and catalytic properties of all of the class II RNA polymerases remain unchanged. Further, the subunit architecture of the predominant form(s) of polymerase II is unaltered. These findings suggest that although new RNA sequences are transcribed during encystment their appearance is not a consequence of extensive alterations in the subunit composition of the major class II RNA polymerase.

DNA-dependent RNA polymerase I from Acanthamoeba castellanii: Comparison of the catalytic properties and subunit architectures of the trophozoite and cyst enzymes

The actively growing cells (trophozoites) of the amoeba Acanthamoeba castellanii were found to contain one major form of class I RNA polymerase. This enzyme was purified to near homogeneity and had a subunit architecture of (182,000) 1, (122,000) 1, (35,000) 2, (20,800) 2, (19,900) 1, (18,100) 1, and (16,400) 2. The subcellular localization and catalytic properties of the enzyme suggest it to be a cognate of the eucaryotic class I polymerases previously reported. When Acanthamoeba castellanii is transferred to a medium lacking nutrients, the cells undergo a cellular differentiation resulting in the formation of metabolically inactive cells (cyst formation). During this process, the rate of ribosomal RNA synthesis decreases, ceasing by the seventh hour after cyst induction. During this time polymerase II and III activity remains constant and the cells are metabolically active. In contrast to this decrease in rRNA synthesis, we found no change in the level of extractable polymerase I activity ( S. Detke and M. R. Paule, 1975, Biochim. Biophys. Acta, 383, 67–77 ) or in the specific activity of the purified enzyme. Furthermore, the catalytic and chromatographic properties and the subunit architecture of the polymerase remain unchanged. These findings suggest that the rate of rRNA synthesis in Acanthamoeba is not controlled by modifying the number or the structure of the polymerase I molecules present in the cell.

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.

Experimental pneumonitis and encephalitis caused by acanthamoeba in mice: pathogenesis and ultrastructural features.

For a more precise definition of the clinicopathological features of experimental acanthamoebic infection in mice, trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga were instilled intranasally into adult white mice. Eight to 20 days after inoculation, severe pulmonary disease developed; one to two days later, neurological signs ensued. On pathologic examination an amebic broncho-pneumonia associated with encephalitis was found. Trophozoites and cysts were seen in lung and brain. Although Naegleria is spread by the olfactory route, cerebral lesions produced by Acanthamoeba might result principally from hematogenous carriage from the lungs. Other differences between infections caused by Naegleria and those caused by Acanthamoeba in mice also exist and serve to emphasize that when natural infections with Acanthamoeba occur, a distinct clinicopathological entity may be produced.

DNA-dependent RNA polymerases from Acanthamoeba castellanii: Properties and levels of activity during encystment

Three DNA-dependent RNA polymerases have been isolated and partially purified from trophozoites of Acanthamoeba castellanii. Separated by DEAE-Sephadex chromatography, they have been designated polymerases, I, IIa and IIb according to their α-amanitin sensitivity and kinetic properties. I is completely insensitive to α-amanitin. IIa and IIb are sensitive to low concentrations (0.1 μg/ml) of α-amanitin; however, in order to achieve 100% inhibition much higher concentrations (130 μg/ml) are needed. Both I and II (a or b) have rather broad ionic strength optima (0.06–0.10 M (NH 4) 2 SO 4). All three prefer denatured over native DNA (I, 4 : 1; II, 2 : 1). Polymerase I utilizes magnesium better than manganese as divalent cation whereas II prefers manganese. When Acanthamoeba is transferred to a medium lacking nutrients, the cells undergo a synchronous differentiation resulting in cyst formation. In general agreement with the decrease in the rate of synthesis of its product (rRNA), the amount of polymerase I decreases relative to the amanitin sensitive polymerase(s). However, the absolute amount of polymerase I does not change. Rather, the levels of the amanitin sensitive enzymes increase during the first 10 h of encystment. Since the overall RNA synthesis rate decreases, these results suggest that the transcription rate is not controlled by specific enzyme levels alone.

Biological Sciences: Effects of Cycloheximide on RNA Polymerase Specific Activity and Encystment in Acanthamoeba castellanii

TROPHOZOITES of the protozoan Acanthamoeba castellanii (Neff) possess at least two distinct RNA polymerase activities which can be separated by DEAE-‘Sephadex’ A-50 chromatography1. These enzymes show optimum activities at different magnesium, manganese, and salt concentrations. Polymerase fraction I is also inhibited by cycloheximide, while polymerase fraction II is affected by rifampin and α-amanitin.