Naegleria actin elicits species-specific antibodies

Actin, the major protein of amebae of Naegleria gruberi, proved to be strongly immunogenic in rabbits. The resulting precipitating antibodies are specific to actin of Naegleria. In a competitive solid-phase radioimmunoassay, these antibodies bound similarly to Naegleria G- and F-actin. Actins from amebae of Acanthamoeba and Dictyostelium, plasmodia of Physarum, sea urchin eggs, and vertebrate muscles gave no competition in the radioimmunoassay. Estimates of the amount of actin in Naegleria amebae ranged from a minimum of 5% of the total cell protein by radioimmunoassay to a maximum of 16% by electrophoresis. The unusual species specificity of these antibodies indicates that Naegleria actin, although conserved in many properties, is different enough to have unique antigenic determinants.     

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.     

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.     

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.     

NACM, A Cytopathogenic Protein from Naegleria gruberi, EGs; Purification, Production of Monoclonal Antibody, and the Immunoidentification of a Product that Develops in NACM-treated Vertebrate Cell Cultures

The story of NACM involves the discovery of a deleterious response of cultured vertebrate cells to a component in cell-free lysates prepared from free-living amebae of the genus Naegleria; hence the acronym NACM derived from Naegleria ameba cytopathogenic material. The cellular reaction is the basis for the biological assay that has been fundamental in the study of the action of NACM in a variety of cell cultures. It also has been used in the determination of the physical characteristics, and to monitor the behavior of NACM during isolation procedures. All findings are compatable with the conclusion that NACM is a 35 Kd protein. Recently, the use of monoclonal antibodies (MAbs) prepared to amebae-derived purified NACM have resulted in visual display of a product that develops exclusively in NACM-treated cells. That cellular product is shown to be related to NACM by its immunostaining reaction with the MAb; the relationship of the MAb with NACM is demonstrated by its ability to neutralize the biological activity of NACM, and as an immunostain, to react with purified fractions of NACM and with whole amebae. The combination of these observations describes a unique set of interactions in which NACM, an amebic component, identified as a protein, has characteristics of an infectious agent when introduced into cultures of avian and mammalian cells.     

Cellular and Environmental Variables Determining Numbers of Flagella in Temperature-shocked Naegleria*

SYNOPSIS Naegleria gruberi amebae normally transform into biflagellated cells. When subjected to high temperatures during flagellate differentiation, populations develop an average of 4–5 flagella/flagellate. Attempts to maximize this phenomenon by altering cellular and environmental variables revealed that: (a) few Naegleria isolates become multiflagellated: strain NB-1 gives the greatest response to heat shocks: (b) temperature is the most critical variable: highest numbers of flagella are obtained only if cells are temperature-shocked at precisely 38.2 ± 0.1 C, then returned to 19–22 C to complete differentiation; (c) although pH alone does not affect numbers of flagella. a pH optimum of 5.5–7.0 exists for temperature-shocked cells; and (d) single cells in microdrops become multiflagellated, but the population response is density-dependent. Optimal conditions are described for growing, washing, and transforming amebae to generate reproducibly highest numbers of flagella.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS OF NAEGLERIA

Flagellates of Naegleria gruberi have an interconnected flagellar apparatus consisting of nucleus, rhizoplast and accessory filaments, basal bodies, and flagella. The structures of these components have been found to be similar to those in other flagellates. The development of methods for obtaining the relatively synchronous transformation of populations of Naegleria amebae into flagellates has permitted a study of the development of the flagellar apparatus. No indications of rhizoplast, basal body, or flagellum structures could be detected in amebae. A basal body appears and assumes a position at the cell surface with its filaments perpendicular to the cell membrane. Axoneme filaments extend from the basal body filaments into a progressive evagination of the cell membrane which becomes the flagellum sheath. Continued elongation of the axoneme filaments leads to differentiation of a fully formed flagellum with a typical "9 + 2" organization, within 10 min after the appearance of basal bodies.     

An Infectious Agent Associated with Amebas of the Genus Naegleria*

A study of amebas of the genera Naegleria, Acanthamoeba, Polysphondylium, and Didymium shows that a cytopathogenic agent that is filterable and passageable is present only in the strains of the Naegleria whether they are obtained free-living from soil samples (N. gruberi) or as pathogens from humans (N. fowleri). The agents obtained from the different Naegleria strains are similar in amount and in their cytopathogenic interaction with chick cultures. The agent has characteristics that distinguish it from the known viruses.     

Rapid disappearance of translatable actin mRNA during cell differentiation in Naegleria

Actin, the major protein of Naegleria gruberi, is selectively not synthesized during the differentiation of amebae to flagellates. When RNA extracted from cells at intervals during differentiation is translated in the wheat germ cell-free system, a major translation product with the electrophoretic mobility of actin is seen to disappear with time during differentiation. This translation product is shown to be actin by its electrophoretic mobility, copolymerization with rabbit actin, peptide map, and immunoprecipitation by antibodies specific to Naegleria actin. Multiple isoforms of actin are synthesized in the cell-free system. Quantitative immunoprecipitation of translation products was employed to measure the relative amount of actin mRNA. Translatable actin mRNA begins to decrease in abundance within 7 min after the initiation of differentiation and thereafter decreases with a half-life of about 25 min. The selective disappearance of this major translatable mRNA provides a favorable opportunity to dissect the rules governing the half-life of a specific mRNA.     

Rapid, Sensitive, and Discriminating Identification of Naegleria spp. by Real-Time PCR and Melting-Curve Analysis

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.     

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.     

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.     

Prevalence of Naegleria fowleri in Environmental Samples from Northern Part of India

Naegleria fowleri the causative agent of Primary Amoebic Meningoencephalitis, is ubiquitously distributed worldwide in various warm aquatic environments and soil habitats. The present study reports on the presence of Naegleria spp. in various water bodies present in Rohtak and Jhajjar district, of state Haryana, India. A total of 107 water reservoirs were screened from summer till autumn (2012 and 2013). In order to isolate Naegleria spp. from the collected water samples, the water samples were filtered and the trapped debris after processing were transferred to non-nutrient agar plates already seeded with lawn culture of Escherichia coli. Out of total 107 water samples, 43 (40%) samples were positive by culture for free living amoeba after incubation for 14 days at 37°C. To identify the isolates, the ITS1, 5.8SrDNA and ITS2 regions were targeted for PCR assay. Out of total 43 positive samples, 37 isolates were positive for Naegleria spp. using genus specific primers and the most frequently isolated species was Naegleria australiensis. Out of 37 Naegleria spp. positive isolates, 1 isolate was positive for Naegleria fowleri. The sequence analysis revealed that the Naegleria fowleri strain belonged to Type 2.     

A Comparative Study of 14 Strains of Naegleria australiensis Demonstrates the Existence of a Highly Virulent Subspecies: N. australiensis italica n. spp.1

Fourteen strains of Naegleria australiensis, including the type strain, were compared for virulence for mice, maximum growth temperature, lectin agglutination, isoenzyme pattern, and total protein banding pattern. Their relation to other species of Naegleria also was compared by immunoelectrophoretic analysis. Strains with high virulence, comparable to that of N. fowleri, were found to be different in concanavalin A agglutination as well as with regard to zymograms and total protein patterns. Although serologically different from N. fowleri and reacting with N. australiensis antiserum in the fluorescent antibody test, these high-virulence strains differed in number of immunoelectrophoretic precipitin bands. Because of these results, the high-virulence strains are considered to be a subspecies of N. australiensis. The low-virulence strains showed minor differences from the type strain. Thus, N. australiensis does not appear to be as homogenous a species as N. fowleri. Pathogenic N. australiensis also seems to be more widespread than previously thought.     

Successful Demonstration of an Elusive Cell Coat in Amebae*

SYNOPSIS. Cell coats were cytochemically demonstrated for the first time in myxamebae of Fuligo septica, Didymium iridis, Dictyostelium discoideum, Cavostelium apophysatum, and amebae of Naegleria gruberi. The stain enhances the cell coats of Physarum polycephalum plasmodia, Ceratiomyxa fruticulosa myxamebae, and Acanthamoeba sp. Cell coats usually unstained by cationic dyes stain intensely with the aid of the new cytochemical protocol utilizing 0.5% Alcian blue in the primary fixative and 0.05% ruthenium red in the secondary fixative.     

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.     

Epidemiology of Free-Living Ameba Infections1

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred world-wide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acanthamoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycobacteria indicate the public health importance of these amebas.     

Restriction Endonuclease Analysis of Mitochondrial DNA as an Aid in the Taxonomy of Naegleria and Vahlkampfia1

ABSTRACT Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

Naegleria: A Research Partner For Cell and Developmental Biology1

ABSTRACT. Organisms in the genus Naegleria offer special opportunities for research in contemporary biology. the dramatic cell differentiation from amebae to flagellates is unique among eukaryotes in the rapidity, synchrony, reproducibility, homogeneity, and accessibility of a major phenotypic change. Environmental signals initiate a progressive signal transduction pathway in which genes are turned on, including those for several calcium-binding proteins, and newly synthesized proteins become localized in newly assembled organelles, including the centriole-like basal bodies, with the overall consequence that the cell changes its shape, motility, and behavior. This essay reviews research opportunities for which Naegleria excels, as well as interesting aspects of its biology that provide challenges for future investigations. Because these organisms alternate between two major eukaryotic motility forms, their phylogenetic position is also provocative. Although there are hints that Naegleria is capable of sexual reproduction in nature, mating has not yet been observed in the laboratory. In order to fully exploit the opportunities offered by this wonderful experimental system we are working to develop means to do genetic manipulation, in particular via DNA-mediated transformation.     

Distribution and possible interrelationships of pathogenic and nonpathogenicAcanthamoeba from aquatic environments

Among the more recently discovered agents of human disease are small, free-living amebae belonging to the generaNaegleria andAcanthamoeba. An overview of the distribution ofAcanthamoeba in recent surveys of the near shore waters of the northeastern United States is presented. There appears to be a particular association between the presence ofAcanthamoeba in marine sediments and the sites of oceanic sludge dumping. Amebae belonging to the genusNaegleria have not been isolated from these marine sediments which routinely yieldedAcanthamoeba. Starch gel electrophoretic analysis of enzymes suggests that some isolates ofAcanthamoeba from oceanic sludge dump-sites are not members of previously recognized pathogenic species.