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?     

Partial Purification and Characterization of Naegleria fowleriβ-Glucosidase1

Naegleria fowleri cells, grown axenically, contain high levels of β-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-β-D-glucopyranoside (4MUGlc) (K_m, 0.9 mM), octyl-β-D-glucoside (K_m, 0.17 mM), and p-nitrophenyl-β-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the β-glucosidase activity appears in the supernatant fraction. The β-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble β-D-galactosidase activity in the Naegleria extract copurifies with the β-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (K_i, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleriaβ-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 Å, and a sedimentation coefficient of 4.2S. The β-glucosidase is not inhibited by conduritol β-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol β-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the β-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.     

Acid and Neutral Hydrolases in Trypanosoma cruzi. Characterization and Assay*

Twelve acid hydrolases, 4 near-neutral hydrolases and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and α-naphthylphosphatase, with optimum pH at ? 6.0; α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0: and arylsulfatase cathepsin D, α-arabinase and α-mannosidase with optimum pH at ? 4.0 α-Glucosidase, gluccse-6-phosphatase and peptidase II had optimum pH at ? 7.0. β-Glycerophcsphatase had a broad pH-activity curve from 4.0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for α-fucosidase, β-xylosidase, β-glucuronidase, elaidate esterase. acid lipase, and alkaline phospho-diesterase.     

Movement of Naegleria fowleri Stimulated by Mammalian Cells In Vitro1

ABSTRACT. Naegleria fowleri amebae, but not those of N. australiensis, N. gruberi, or N. lovaniensis, demonstrated enhanced motility when placed in proximity to mammalian cells. Amebae of nonpathogenic species of Naegleria, however, were more motile in cell culture medium than the amebae of N. fowleri. The locomotory response of highly pathogenic mouse-passaged N. fowleri amebae to nerve cells was greater than axenically cultured amebae. The enhanced mobility elicited by whole nerve cells or disrupted nerve cells was not directed migration but chemokinetic. Naegleria fowleri responded to disrupted neuroblastoma cells more vigorously than to disrupted African green monkey kidney (Vero) cells.     

Hydrolytic Enzymes of Euglena gracilis: Characterization and Activity as a Function of Culture Age and Carbon Deprivation*†

SYNOPSIS. Optimal assay conditions are described for 8 hydrolases of Euglena gracilis var. bacillaris, SM-L1 (streptomycinbleached) strain, 7 of which have an acid pH-optimum. Acid phosphatase, β-galactosidase, β-glucosidase, β-fucosidase, cathepsin D, RNase, DNase, and an esterase are active in cell homogenates. Amylase has very low activity, and β-glucuronidase, arylsulfatase, β, N-acetyl-glucosaminidase, α-fucosidase, and α- and β-mannosidase are inactive. Hydrolase activity increases as a culture proceeds from the midexponential to the late stationary-phase of growth, being most pronounced in the case of β-glucosidase. In cultures deprived of a utilizable carbon source, the specific activities of the hydrolases (per mg total protein or dry weight) increase. When expressed on a per cell basis, however, the activities of DNase decrease while those of β-galactosidase, cathepsin D, and RNase increase. The hydrolases appear to be involved in the adaptation of Euglena to the metabolic demands imposed by different conditions of growth.     

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts. International journal for Parasitology4: 139–142. Cysts of 4 strains of non-pathogenic Naegleria gruberi and 5 strains of pathogenic Naegleria fowleri were examined in the scanning electron microscope. Excystment of the Naegleria gruberi amoebae occurred via preformed exit pores in the cyst wall. Similar structures were not found in the cysts of Naegleria fowleri, and excystment occurred by rupture of the cyst wall. The sequence of cyst wall rupture is illustrated for one of the pathogenic strains.     

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.     

Antiparasitic Activity of Auranofin against Pathogenic Naegleria fowleri

We report that the gold containing antirheumatoid drug auranofin is amoebicidal against human pathogenic Naegleria fowleri. Treatment of N. fowleri cultures at biologically relevant concentrations of 0.75–3.0 μg/ml auranofin reduced amoeba counts, metabolic activity, and increased cell permeability. These results suggest that the addition of auranofin may benefit the treatment of N. fowleri‐infected patients afflicted by the rapidly fatal disease primary amoebic meningoencephalitis.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

Lysosomes in Tetrahymena pyriformis. I. Some properties and lysosomal localization of acid hydrolases

In homogenates of Tetrahymena pyriformis, five hydrolases — phosphatase, ribonuclease, deoxyribonuclease, proteinase, amylase — with acid pH optima were found. Over 75% of their activity is sedimentable with a centrifugal force of 250,000 g. min. Only 17% of the acid phosphatase and ribonuclease is active when assayed in the presence of 0.25 M sucrose at 0°. Exposure to a lowered osmotic pressure, freezing and thawing, and incubation at temperatures over 0° result in activation of the latent phosphatase and ribonuclease. After isopycnic centrifugation in a sucrose density gradient the hydrolases show a broad distribution which differs greatly from those of enzymes associated with mitochondria (succinate dehydrogenase) or with peroxisomes (catalase). The results are interpreted as evidence that the five acid hydrolases studied are localized in lysosomes which represent a distinct population of subcellular particles in Tetrahymena.     

Acid-active neuraminidases in the growht media from cultures of pathogenic Naegleria fowleri and in sonicates of rabbit alveolar macrophages

Using bovine mucin and isolated human myelin as source of sialic acid, we demonstrate the presence of neuraminidase activities in the growth media of pathogenic, but not nonpathogenic, Naegleria sp. and in sonicates of rabbit alveolar macrophages. Neuraminidase activity was maximal at pH 4.5 and 5.0, and the specific activity for sialic release was up to 13-fold greater with mucin that with human myelin. Activity in the growth media from cultures of pathogenic Naegleria fowleri was ion-independent, while that of macrophage sonicates required divalent cation; optimal activity was noted with 2.5 mM Zn², while Mg²⁺ and Mn²⁺ supported activity to a lesser extent. Such acid-active neuraminidases may contribute to the reported glycolipid alterations associated with the demyelinating diseases.     

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.     

The Mitochondrial Genome and a 60‐kb Nuclear DNA Segment from Naegleria fowleri,the Causative Agent of Primary Amoebic Meningoencephalitis

Naegleria fowleri is a unicellular eukaryote causing primary amoebic meningoencephalitis, a neuropathic disease killing 99% of those infected, usually within 7–14 days. Naegleria fowleri is found globally in regions including the US and Australia. The genome of the related nonpathogenic species Naegleria gruberi has been sequenced, but the genetic basis for N. fowleri pathogenicity is unclear. To generate such insight, we sequenced and assembled the mitochondrial genome and a 60‐kb segment of nuclear genome from N. fowleri. The mitochondrial genome is highly similar to its counterpart in N. gruberi in gene complement and organization, while distinct lack of synteny is observed for the nuclear segments. Even in this short (60‐kb) segment, we identified examples of potential factors for pathogenesis, including ten novel N. fowleri‐specific genes. We also identified a homolog of cathepsin B; proteases proposed to be involved in the pathogenesis of diverse eukaryotic pathogens, including N. fowleri. Finally, we demonstrate a likely case of horizontal gene transfer between N. fowleri and two unrelated amoebae, one of which causes granulomatous amoebic encephalitis. This initial look into the N. fowleri nuclear genome has revealed several examples of potential pathogenesis factors, improving our understanding of a neglected pathogen of increasing global importance.     

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.     

Identification of Peptidases in Highly Pathogenic vs. Weakly Pathogenic Naegleria fowleri Amebae

Naegleria fowleri, a free‐living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse‐passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba‐CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.     

Partial purification and characterization of Naegleria fowleri beta-glucosidase

Naegleria fowleri cells, grown axenically, contain high levels of beta-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGlc) (Km, 0.9 mM), octyl-beta-D-glucoside (Km, 0.17 mM), and p-nitrophenyl-beta-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the beta-glucosidase activity appears in the supernatant fraction. The beta-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble beta-D-galactosidase activity in the Naegleria extract copurifies with the beta-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (Ki, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleria beta-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 A, and a sedimentation coefficient of 4.2S. The beta-glucosidase is not inhibited by conduritol beta-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol beta-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the beta-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.     

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV‐2 Microglial Cells

Naegleria fowleri, a free‐living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV‐2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV‐2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL‐1α and TNF‐α. NfESP‐induced IL‐1α and TNF‐α expression in BV‐2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP‐induced IL‐1α and TNF‐α production in BV‐2 cells were effectively downregulated by inhibition of NF‐kB and AP‐1. These results collectively suggest that NfESP stimulates BV‐2 cells to release IL‐1α and TNF‐α via NF‐kB‐ and AP‐1‐dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.     

Comparative Study of Six Strains of Naegleria with Special Reference to Nonpathogenic Variants of Naegleria fowleri*

SYNOPSIS. Naegleria fowleri strains HB-1 and KUL, pathogenic for humans, Naegleria gruberi strain 1518/1e, and 3 strains (Vm1, LvH₁, and LvH₂) of Naegleria isolated from a body of water polluted with thermal effluents were compared in an attempt at specific identifications of the latter strains. The 3 environmental isolates were morphologically almost identical with N. fowleri and had almost the same temperature tolerance, although at 37 and 42 C the growth rates of LvH₁ and LvH₂ were higher than those of the human pathogen, N. fowleri, and of isolate Vm1, which was pathogenic for mice. Serologic examinations by indirect fluorescent antibody method revealed a very close relationship of the new isolates with the human pathogens. While Vm1 was indistinguishable from N. fowleri, LvH₁ and LvH₂ were not, when cross-absorbed antisera were used. Of all the strains examined, only the 2 LvH isolates were not inhibited by amphotericin B, while only N. gruberi was not inhibited by fumagillin. The cytopathic effect in Vero cell cultures suggested that the LvH strains could have a certain degree of virulence, although this was not confirmed by intranasal and intracerebral inoculations of mice. The cytopathic effects of the human pathogens and of the isolate pathogenic for mice were related to their virulence for mice. It is concluded that there exists an intermediate form between N. gruberi and N. fowleri, with a strong relationship to the latter species. We refer to such strains as nonpathogenic variants of N. fowleri. Further research is needed to reveal their place in the taxonomy.     

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.