Subcellular Distribution of Hydrolases in Naegleria fowleri1

The presence and particle association of various hydrolytic enzymes in Naegleria fowleri has been studied in whole cell extracts of trophozoites in an effort to establish authentic markers for surface membrane and lysosomal components. Evidence from the experiments reported here indicates that in N. fowleri a) acid proteinase, N-acetylglucosaminidase, and acid phosphatase are associated with cytoplasmic granules closely resembling lysosomes; b) 5'-nucleotidase is associated with the surface membrane, probably on the external surface; c) aspartate aminotransferase is associated with mitochondria; d) a-D-glucosidase and an aminopeptidase have bimodal distributions, activity being associated with both the surface membrane and lysosomal particles.     

Amebostomes of Naegleria fowleri1

The strain of ameba, culture incubation temperature, and phase of ameba growth affected the number of amebostomes present on amebae of Naegleria fowleri. Serial passage of N. fowleri through mice decreased the average number of amebostomes. Amebostomes were shown to be functional by their ability to engulf yeast cells.     

Serology of Naegleria fowleri and Naegleria lovaniensis in a Hospital Survey1

An avidin-biotin horseradish peroxidase method was used to detect antibodies to Naegleria fowleri and N. lovaniensis in human serum samples. Antibodies were detected in 101 specimens from 115 hospital patients ranging in age from 15 to 98 years. Class-specific anti-immunoglobulins identified antibodies as IgG and IgM. IgG antibody titers to both species ranged from 1:20 to 1:640. Seven of 15 serum samples collected from newborn infants also demonstrated IgG antibodies to these organisms with a titer range of 1:20 to 1:80. The immunoperoxidase test and Western blot analysis of selected serum samples demonstrated a close similarity in serological results between N. fowleri and N. lovaniensis.     

Characterization of a neutral aminoacyl-peptide hydrolase from Naegleria fowleri

An intracellular alpha-aminoacyl-peptide hydrolase (EC 3.4.11.-) from Naegleria fowleri nN68 (ATCC 30894) has been characterized. The enzyme preparation hydrolyzed phenylalanyl-, tyrosyl-, leucyl-, arginyl-, alanyl-, tryptophanyl-, histidyl-, methionyl-, and lysyl-naphthylamide but not benzoylleucyl-, leucylglycyl-, glycylprolylleucyl-, glycyl-, threonyl-, aspartyl-, or glutamyl-naphthylamide. The aminopeptidase activity was inhibited by the cysteine-protease inhibitors--hydroxymercuribenzoate, chloromercurisulfate, and iodoacetate--by the aminopeptidase inhibitors--bestatin and trans-epoxysuccinyl-leucyl-agmatine--by an inhibitor of soluble alanyl aminopeptidase EC 3.4.11.14, puromycin, and by the metalloprotease inhibitor, o-phenanthroline. The exopeptidase activity was not inhibited by the chelator, ethylenediaminetetraacetate, or the serine-protease inhibitor, phenylmethylsulfonylfluoride. The pH optimum of the exopeptidase was between 7.0 and 8.0. Enzyme activity was stable at 55 degrees C for 30 min, but all activity was lost after 15 min at 80 degrees C. Enzyme activity was inhibited by 100 microM HgCl2 and CdCl2 but not by 1 mM CoCl2, CuCl2, MnCl2, NiCl2, FeCl3, or ZnCl2. Enzyme activity was inhibited by 0.1% sodium dodecyl sulfate but not by 0.2% Brij 35, Tween 20, Tween 80, or Triton X-100.     

Conditions for Maximum Enflagellation in Naegleria fowleri1

ABSTRACT. Ameba to flagellate transformation in Naegleria fowleri (Lovell strain) was affected by growth temperature, phase of growth, strain of ameba, culture agitation, enflagellation temperature, enflagellation diluent, and cell concentration. Amebae transformed best when they were grown without agitation and enflagellated with agitation. Regardless of growth temperature (23°, 30°, 37°, and 42°C were tested), amebae transformed best at 37°C. Enflagellation was greatest for cells harvested between 24 h (mid-exponential) and 84 h (late stationary) of growth.     

Chemotaxis by Naegleria fowleri for Bacteria1

Naegleria fowleri amebae demonstrated a chemotactic and chemokinetic response toward live cells and extracts of Escherichia coli and other bacterial species when experiments were performed using a blind-well chemotaxis chamber. The peptide N-formyl-methionyl-leucyl-phenylalanine acted as a chemokinetic rather than a chemotactic factor for N. fowleri amebae. Competition experiments in which nerve cell extracts or bacteria were placed on either side of the filter in chemotaxis chambers resulted in increased movement towards bacteria. A scanning electron microscopy study of the interaction of N. fowleri with different bacterial species confirmed that when the amebae were near ingestible bacteria they moved toward the bacteria by pseudopod formation. Naegleria fowleri appeared to respond to bacteria by three interrelated but distinct processes: (a) chemokinesis, (b) chemotaxis, and (c) formation of food cups.     

Amebostomes of Naegleria fowleri

The strain of ameba, culture incubation temperature, and phase of ameba growth affected the number of amebostomes present on amebae of Naegleria fowleri. Serial passage of N. fowleri through mice decreased the average number of amebostomes. Amebostomes were shown to be functional by their ability to engulf yeast cells.     

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.     

Cytolytic Activity of Naegleria fowleri Cell-free Extract1

ABSTRACT. The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30–60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by ⁵¹Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50°C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing ⁵¹Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Intranasal Immunization of Mice Against Naegleria fowleri1

ABSTRACT. The purpose of this research was to determine whether mice could be protected from lethal challenge with Naegleria fowleri by prior intranasal exposure to pathogenic and nonpathogenic Naegleria. Mortality ranged from 0 to 100% for mice inoculated intranasally (i.n.) with 5 × 10³ amebae of 13 human isolates of N. fowleri. Mice were immunized and challenged i.n. using live amebae of strains of low, medium, and high virulence. The greatest protection against lethal challenge was afforded by three immunizing doses of 10³ amebae per dose of the strain of medium virulence. Nonpathogenic N. gruberi also was used to immunize mice i.n. against lethal challenge with N. fowleri. Protection was greater following immunization with N. gruberi than it was after immunization with N. fowleri, suggesting that nonpathogenic N. gruberi may be a better immunogen in protecting mice against lethal naeglerial challenge.     

Demonstration of various acid hydrolases and preliminary characterization of acid phosphatase in Naegleria fowleri

Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include beta-glucosidase, beta-galactosidase, beta-fucosidase, alpha-mannosidase, hexosaminidase, arylsulfatase A, and beta-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly "acid" hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: pH optima, 5.5; Km (4-methylumbelliferyl phosphate), 0.60 mM; molecular weight (estimated by gel filtration chromatography), 92,000; inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?     

Characterization of proteins in flagellates and growing amebae of Naegleria fowleri.

Polypeptides of whole-cell extracts of Naegleria fowleri flagellates and growing amebae were resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [35S]methionine-labeled polypeptides of amebae and flagellates were analyzed by two dimensional densitometry to determine whether there were correlations between intracellular concentration of a protein and subunit size or charge. The majority of the polypeptides of amebae and flagellates had molecular sizes in the range of 20 to 60 kilodaltons. The radioactivity per polypeptide species in the size range of 20 to 60 kilodaltons was greater in amebae than in flagellates. The greatest number of polypeptides detected in amebae and flagellates was in the isoelectric focusing range of pH 6 to 7. The radioactivity per polypeptide species in the isoelectric focusing gradient below 6.3 was greater in amebae than in flagellates. Polypeptides in the size range of 20 to 60 kilodaltons had a median isoelectric point below pI 6.3, whereas those larger than 60 kilodaltons had a median pI value above 6.3. These data indicated that molecular size and charge were not entirely independent variables and that the size and charge of a polypeptide might have an important influence in determining its intracellular concentration in both amebae and flagellates. Autoradiograms were also compared so that changes in intracellular protein complement and concentrations occurring during differentiation could be recognized. The relative amounts of a limited number of polypeptides increased markedly, and others decreased markedly, during enflagellation.     

Characterization of the Proteins of Naegleria fowleri: Relationships between Subunit Size and Charge1

Proteins of whole cell extracts of Naegleria fowleri, precipitated with acetone, have been resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [³⁵S]-methionine-labeled polypeptides were scanned and analyzed by a computer-assisted program in order to determine whether there were correlations between selected attributes of proteins (e.g., subunit size and charge). The majority of the polypeptides had molecular sizes within the range of 20–60 kilodaltons. The mean amount of polypeptide was less for those with molecular sizes between 20 and 45 kilodaltons than for those larger than 45 kilodaltons. The mean amount of polypeptide was greater in the isoelectric focusing range of pH 5–6 than in the range of pH 6–7. Polypeptides in the size range of 20–40 kilodaltons had a median isoelectric point of 6.1, whereas polypeptides in the size range of 40–80 kilodaltons had a median pI of 5.6. Our data indicated that molecular size and charge were not entirely independent variables, and that the composition of a polypeptide might have an important influence on its steady state level in N. fowleri.     

Serology of Naegleria fowleri and Naegleria lovaniensis in a hospital survey

An avidin-biotin horseradish peroxidase method was used to detect antibodies to Naegleria fowleri and N. lovaniensis in human serum samples. Antibodies were detected in 101 specimens from 115 hospital patients ranging in age from 15 to 98 years. Class-specific anti-immunoglobulins identified antibodies as IgG and IgM. IgG antibody titers to both species ranged from 1:20 to 1:640. Seven of 15 serum samples collected from newborn infants also demonstrated IgG antibodies to these organisms with a titer range of 1:20 to 1:80. The immunoperoxidase test and Western blot analysis of selected serum samples demonstrated a close similarity in serological results between N. fowleri and N. lovaniensis.     

Microfilaments in Naegleria fowleri amoebae.

Examination by electron microscopy has revealed 2 types of microfilament in the cytoplasm of 3 strains of axenically grown Naegleria fowleri amoebae. Thin, actin-like microfilaments 5-7 nm in diameter are randomly oriented in the nonmotile amoebae, and are concentrated near the plasma membrane. In the actively motile amoebae these microfilaments aggregate to form colateral bundles in close proximity to the plasma membrane. Thick, myosin-like microfilaments 17-19 nm in diameter also occur in the amoebae cytoplasm. The significance of these 2 kinds of microfilament in amoeboid motion is discussed.     

Ultrastructure of Naegleria fowleri enflagellation.

Amoebae of Naegleria fowleri nN68 became elongated flagellated cells 150 to 180 min after subculture to non-nutrient buffer. N. fowleri NF69 did not become elongated or flagellated under these conditions. Electron microscopic examination of N. fowleri confirmed that it is a typical eucaryotic protist with a distinct nuclear envelope and prominent nucleolus, numerous vacuoles and cytoplasmic inclusions, pleomorphic mitochondria, and some rough endoplasmic reticulum. During incubation in non-nutrient buffer, both strains lost ultraviolet-absorbing material to the medium, and the number of vacuoles decreased. In strain nN68, basal bodies, a rootlet, and flagella are formed quickly after an initial lag of 90 min. Initially, the rootlet is not associated with the nucleus but they become associated subsequent at the leading end of the elongated cell. In elongated cells, the rootlet lies in a furrow or groove extending the length of the nucleus. Flagella of N. fowleri nN68 exhibit the typical 9 + 2 arrangement of filaments and are surrounded by a sheath which is continuous with the plasma membrane. The enflagellation process in N. fowleri can be manipulated reproducibly.     

Subcellular distribution of hydrolases in Naegleria fowleri

The presence and particle association of various hydrolytic enzymes in Naegleria fowleri has been studied in whole cell extracts of trophozoites in an effort to establish authentic markers for surface membrane and lysosomal components. Evidence from the experiments reported here indicates that in N. fowleri a) acid proteinase, N-acetylglucosaminidase, and acid phosphatase are associated with cytoplasmic granules closely resembling lysosomes; b) 5'-nucleotidase is associated with the surface membrane, probably on the external surface; c) aspartate aminotransferase is associated with mitochondria; d) alpha-D-glucosidase and an aminopeptidase have bimodal distributions, activity being associated with both the surface membrane and lysosomal particles.     

Conditions for maximum enflagellation in Naegleria fowleri

Ameba to flagellate transformation in Naegleria fowleri (Lovell strain) was affected by growth temperature, phase of growth, strain of ameba, culture agitation, enflagellation temperature, enflagellation diluent, and cell concentration. Amebae transformed best when they were grown without agitation and enflagellated with agitation. Regardless of growth temperature (23 degrees, 30 degrees, 37 degrees, and 42 degrees C were tested), amebae transformed best at 37 degrees C. Enflagellation was greatest for cells harvested between 24 h (mid-exponential) and 84 h (late stationary) of growth.     

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.