Naegleria gruberi metabolism

The completion of the genome project for Naegleria gruberi provides a unique insight into the metabolic capacities of an organism, for which there is an almost complete lack of experimental data. The metabolism of Naegleria seems to be extremely versatile, as can be expected for a free-living amoeboflagellate, but although considered to be fully aerobic, its genome also predicts important anaerobic traits. Other predictions are that carbohydrates are oxidised to carbon dioxide and water when oxygen is not limiting and that in the absence of oxygen the end-products will be succinate, acetate and minor quantities of ethanol and d-lactate. The hybrid mitochondrion/hydrogenosome has both cytochromes and an [Fe] hydrogenase, but seems to lack pyruvate-ferredoxin oxidoreductase. Genomic information also provides the possibility to identify drugs with a possible mode of action in the fatal primary amoebic meningoencephalitis caused by the closely related opportunistic pathogen Naegleria fowleri.     

Naegleria fowleri and Naegleria gruberi 20S proteasome: identification and characterization

The Naegleria are ubiquitous free-living amoebae and are characterized by the presence of three phases in their biological cycle: trophozoite, cyst and flagellate. Of this genus, only Naegleria fowleri has been reported as pathogenic to humans. The proteasome is a multi-catalytic complex and is considered to be the most important structure responsible for the degradation of intracellular proteins. This structure is related to the maintenance of cellular homeostasis and, in pathogenic microorganisms, to the modulation of their virulence. Until now, the proteasome and its function have not been described for the Naegleria genus. In the current study, using bioinformatic analysis, protein sequences homologous to those reported for the subunits of the 20S proteasome in other organisms were found, and virtual modelling was used to determine their three-dimensional structure. The presence of structural and catalytic subunits of the 20S proteasome was detected by Western and dot blot assays. Its localization was observed by immunofluorescence microscopy to be mainly in the cytoplasm, and a leading role of the chymotrypsin-like catalytic activity was determined using fluorogenic peptidase assays and specific proteasome inhibitors. Finally, the role of the 20S proteasome in the proliferation and differentiation of Naegleria genus trophozoites was demonstrated.     

Acriflavin Inhibition of Morphogenesis in Naegleria gruberi*

SYNOPSIS. When amoeboid organisms of Naegleria gruberi are treated with acriflavin at concentrations under 70 μg/ml, they still can form flagellates. Acriflavin between 70 μg/ml and 110 μg/ml increasingly inhibits morphogenesis. The effect of acriflavin is specific for the morphogenetic system since amoeboid organisms treated with acriflavin between 110 and 200 μg/ml are not otherwise inhibited and behave like the controls.     

Ribonucleoprotein-Containing Vesicles in Cysts of Naegleria gruberi*

SYNOPSIS. Ultraviolet microscopy and electron microscope autoradiography were used to study ribonucleoprotein in cysts of Naegleria gruberi. The absorption maximum for cysts is at 265 nm with little detectable absorption occurring at 295 nm. Pre-cystic trophozoites absorb less strongly than the cysts at 265 mm. Acridine orange staining indicated concentrations of ribose nucleic acid or ribonucleoprotein in the cytoplasm of young cysts. The dye stained discrete vesicles in the cytoplasm. Tritiated uridine and tritiated proline were used to follow changes in RN-protein at encystment. Label was incorporated into vesicles filled with ribosome-like particles. These are presumably the sites of acridine orange staining. Relatively little label was associated with the cyst cytoplasmic matrix; most of the silver grains lay over the nucleus and cytoplasmic organelles. The vesicles are believed to represent autophagosomal-type vacuoles with the contents derived from breakdown of organelles such as mitochondria. The path of label into the vesicles is via lysis of labeled cytoplasmic organelles. The RN-protein vesicles of Naegleria gruberi cysts are compared to the chromatoid bodies of Entamoeba invadens. It is concluded that, tho differences in detail are present, the role of the structures in the cysts is probably the same. They are a ready source of amino acids and ribosomes in a dedifferentiated or pool state to be used for synthetic reactions that accompany resumption of trophic existence.     

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.     

Nutritional Study of Three Strains of Naegleria gruberi*

Naegleria gruberi strains cloned from amebas isolated from a Vero cell culture (“TS”), a sewer drainage ditch (“PD”), and an established laboratory line (“S”) were morphologically identical except for differences in size and flagellate transforming ability. Cultivation on a Trypticase-yeast extract-glucose medium (“TYG”) fortified with autoclaved E. coli resulted in increased cell size of 2 strains. Differences also were noted in growth rates and optimal growth temperatures. The autoclaved E. coli in TYG medium was replaceable with serum only for strains TS and PD. A basal salts medium + autoclaved E. coli supported growth of all 3 strains, but the basal salts medium + serum would not support growth of any of the strains.     

Intranuclear Virus-like Bodies in the Amoeboflagellate Naegleria gruberi*

SYNOPSIS. Intranuclear virus-like bodies were seen in cultures of the EG strain of Naegleria gruberi from soil. Introduction of these virus-like particles into cultures seemed to coincide with use of chicken embryo extract as a supplement in culture media used to maintain axenic amoebae in the laboratory; the appearance of the virus-like units is triggered by transfer of axenic lines of N. gruberi EG into monobacterial culture medium. The particles, ～ 100 nm in diameter, are mainly restricted to the nucleus of the cell. Passage of particles from the nucleoplasm into the cytoplasm is suggested by theit association with tubular projections from the nuclear membrane, and particles have been seen in the cytoplasm of the amoebae. The virus-like bodies resemble reovirus.     

Differentiation-Dependent Decline of DNA Synthetic Activities in Naegleria gruberi

SYNOPSIS. DNA of Naegleria gruberi strain NEG, grown in axenic culture, forms a band at a density of 1.6912 in CsCl gradient and has a GC content of 31.8%. Incorporation of [³H]thymidine into DNA is much reduced in differentiating Naegleria immediately after the stimulation to transforms, primarily because of the reduction in thymidine uptake by differentiating cells. In addition, there is a marked decrease in the rate of incorporation of [³H]thymidine and [³H]uracil into DNA at from 45 to 60 min after the stimulation for differentiation. This decrease in the rate of precursor incorporation into DNA appears to be due to the differentiation-dependent cessation of nuclear DNA synthesis. The differentiated phenotype (the flagellate) emerges at ∼ 70 min after the stimulation, and over 90% of the population differentiates within the next 30 min. Synthesis of mitochondrial DNA is detectable until 190 min after the stimulation. Since the S phase of Naegleria lasts ∼ 180 min, some cells in the population must cease synthesizing nuclear DNA in the middle of the S phase.     

Comparison of Naegleria fowleri and Naegleria gruberi Cultivated in the Same Nutrient Medium1

The human pathogenic amoeboflagellate Naegleria fowleri and the nonpathogenic species N. gruberi can be cultivated axenically but usually in different media. Naegleria fowleri 6088 has been adapted to grow in Balamuth H-4 medium, usually used to propagate N. gruberi nB81. and nB81 has been adapted to grow in supplemented Nelson's medium, usually used to propagate N. fowleri. N. gruberi nB81. grown in either medium, enflagellated 135 to 150 min after subculture to non-nutrient amoeba saline, whereas 6088 required 225 min. Naegleria gruberi nB81 grown in either medium was agglutinated by 100 ug concanavalin A/ml, whereas N. fowleri 6088 was not. Naegleria fowleri and N. gruberi grown in Nelson's medium became rounded to a greater extent upon chilling at 5° C and remained rounded longer than Naegleria grown in Balamuth medium. The specificity of the surface antigens was an inherent characteristic of each species and not dependent upon the propagating medium. but Naegleria grown in Nelson's medium was agglutinated more reproducibly and more effectively by antiserum. N. gruberi was somewhat more resistant to acriflavine, actinomycin D, cycloheximide, or tetracycline than N. fowleri, regardless of the culture medium. Naegleria fowleri 6088 grown in Nelson's medium, however, was more resistant to actinomycin D, daunomycin. mithramycin. sulfamethoxazole, or tyrocidine than 6088 grown in Balamuth medium. There are limitations on the validity of comparisons of N. fowleri and N. gruberi based upon cultures grown in different media.     

Preparation and Properties of Mitochondria from Naegleria gruberi*

Whole cell respiration rates were measured polarographically for Naegleria gruberi during growth in agitated cultures. Log growth phase amebae consumed 80 ng atoms O/min/mg cell protein. At stationary phase, respiration rate decreased 4–fold. Intact mitochondria were isolated from N. gruberi and their oxidative and phosphorylative capacities were studied polarographically. As with the mammalian system, the mitochondria oxidized succinate and NAD-linked substrates, but unlike rat liver mitochondria, those from the protozoan rapidly oxidized citrate and NADH. The rates of substrate oxidation were ADP-dependent, with ADP:O ratios equalling ? 2.8 for NAD-linked substrates and ? 2.2 for succinate. The respiratory control ratios. 2 to 4 for 11 substrates, were dependent on Pi, Mg²⁺, and serum albumin. Potassium cyanide, azide, malonale, and rotenone inhibited electron transport the same way as that of the mammalian system: however, amytal inhibited both glutamate and succinate respiration. Pentachlorophenol, DNP, and bilirubin uncoupled oxidation from phosphorylation. Difference spectra of oxidized and dithionite-reduced mitochondria had distinct absorption bands of flavins and of c-, b-, and α-type cytochromes.     

Electrophoretic Karyotype and Linkage Groups of the Amoeboflagellate Naegleria gruberi

ABSTRACT. We have constructed a molecular karyotype for two strains of Naegleria gruberi using pulsed field gel electrophoresis. Each strain has about 23 chromosomes, considerably more than any previous estimate. These chromosomes range in size from 400 kilobasepairs to over 2,000 kilobasepairs. In Naegleria , construction of the DNA karyotype depends on assessment of the anomalous electrophoretic mobility of the circular ribosomal RNA genes. We have determined the chromosomal locations of an identified unique gene (flagellar calmodulin) and four identified multigene families (α- and β-tubulin, actin, ubiquitin), as well as three differentially expressed genes of unknown functions. The ca. 12 actin genes are dispersed over at least seven chromosomes, whereas the majority of the more than eight α-tubulin genes are confined to a single chromosome. The ubiquitin genes are found on five chromosomes in one strain and seven in the other and the β-tubulin genes are on three or four. Our observations provide a foundation for molecular genetic studies in this organism.     

Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.     

Population Size,Clones and Morphogenesis in Naegleria gruberi*

SYNOPSIS. The size of the population of Naegleria gruberi at the time the amoebae are offered the opportunity to become flagellated is not a critical factor in the morphogenesis of this organism. Small populations (1-15 cells) readily become flagellated. Small populations (1-5 cells) washed several times also become flagellated. Clonal populations (25) have been established. All clones yield flagellates under the usual conditions. It is suggested that the physiological state of the amoebae is a factor in determining the number of cells that will undergo morphogenesis at any given time.