Sucker-like structures on the pathogenic amoeba Naegleria fowleri.

Using scanning electron microscopy, we observed sucker-like structures on amoebae of 13 human isolates of Naegleria fowleri. The number of suckers per amoeba seemed to vary according to the virulence of the strain. We propose the term amoebastome to describe this unique sucker-like structure of N. fowleri.     

Pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri

The free-living amoeboflagellate and potential human pathogen Naegleria fowleri causes the often fatal disease primary amoebic meningoencephalitis. The molecular repertoire responsible for the cytolytic and tissue-destructive activity of this amoeboid protozoon is largely unknown. We isolated two pore-forming polypeptides from extracts of highly virulent trophozoites of N. fowleri by measuring their membrane-permeabilizing activity. N-terminal sequencing and subsequent molecular cloning yielded the complete primary structures and revealed that the two polypeptides are isoforms. Both polypeptides share similar structural properties with antimicrobial and cytolytic polypeptides of the protozoon Entamoeba histolytica (amoebapores) and of cytotoxic natural killer (NK) and T cells of human (granulysin) and pig (NK-lysin), all characterized by a structure of amphipathic alpha-helices and an invariant framework of cysteine residues involved in disulfide bonds. In contrast to the aforementioned proteins, the Naegleria polypeptides both are processed from large precursor molecules containing additional isoforms of substantial sequence divergence. Moreover, biochemical characterization of the isolated polypeptides in combination with mass determination showed that they are N-glycosylated and variably processed at the C terminus. The biological activity of the purified polypeptides of Naegleria was examined toward human cells and bacteria, and it was found that these factors, named naegleriapores, are active against both types of target cells, which is in good agreement with their proposed biological role as a broad-spectrum effector molecule.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

Molecular and physiological differentiation between pathogenic and nonpathogenic Acanthamoeba

In this study, 14 isolates of Acanthamoeba from both clinical and environmental sources belonging to seven different species were assayed for tolerance of high osmotic pressure, temperature tolerance, extracellular proteases, and cytopathic effects (CPE) on immortalized rabbit corneal epithelial cells. On the basis of the results, amoeba isolates were divided into pathogenic and nonpathogenic groups. Ribosomal DNA sequencing was performed on these isolates. Phylogenetic relationships revealed that all the pathogenic strains tested clustered together as one group, while nonpathogenic strains clustered into other groups. Sequence comparisons with previously published sequences determined that among the six new pathogenic isolates used in this study, five belong to T4 genotype and one to T11. This is the first report of a T11 genotype being found in Acanthamoeba keratitis. Received: 1 November 2001 / Accepted: 31 December 2001     

Starch gel electrophoresis: an effective method for separation of pathogenic and nonpathogenic Naegleria strains

Isoenzyme electrophoresis of 7 different enzyme systems was used to compare 24 strains of Naegleria fowleri and 6 strains of N. gruberi. The 30 strains could be grouped into 4 distinct categories based upon zymogram patterns. No interstrain band variation in all enzyme systems was demonstrated in pathogenic strains of N. fowleri. Three nonpathogenic high temperature-tolerant strains of Naegleria had similar zymograms. Four of the 5 remaining nonpathogenic Naegleria strains had no interstrain band variation. Based upon zymograms, the 22 pathogenic strains constitute a homogenous species. Similarly the high temperature-tolerant nonpathogenic strains formed a cohesive group. The remaining nonpathogenic strains could be separated into 2 groups.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

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.     

Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing

Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas.     

A biochemical comparison of proteases from pathogenic naegleria fowleri and non-pathogenic Naegleria gruberi

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis (PAM). Proteases have been suggested to be involved in tissue invasion and destruction during infection. We analyzed and compared the complete protease profiles of total crude extract and conditioned medium of both pathogenic N. fowleri and non-pathogenic Naegleria gruberi trophozoites. Using SDS-PAGE, we found differences in the number and molecular weight of proteolytic bands between the two strains. The proteases showed optimal activity at pH 7.0 and 35 degrees C for both strains. Inhibition assays showed that the main proteolytic activity in both strains is due to cysteine proteases although serine proteases were also detected. Both N. fowleri and N. gruberi have a variety of different protease activities at different pH levels and temperatures. These proteases may allow the amoebae to acquire nutrients from different sources, including those from the host. Although, the role of the amoebic proteases in the pathogenesis of PAM is not clearly defined, it seems that proteases and other molecules of the parasite as well as those from the host, could be participating in the damage to the human central nervous system.     

Decreasing effect of an anti-Nfa1 polyclonal antibody on the in vitro cytotoxicity of pathogenic Naegleria fowleri

The nfa1 gene was cloned from a cDNA library of pathogenic Naegleria fowleri by immunoscreening; it consisted of 360 bp and produced a 13.1 kDa recombinant protein (rNfa1) that showed the pseudopodia-specific localization by immunocytochemistry in the previous study. Based on the idea that the pseudopodia-specific Nfa1 protein mentioned above seems to be involved in the pathogenicity of N. fowleri, we observed the effect of an anti-Nfa1 antibody on the proliferation of N. fowleri trophozoites and the cytotoxicity of N. fowleri trophozoites on the target cells. The proliferation of N. fowleri trophozoites was inhibited after being treated with an anti-Nfa1 polyclonal antibody in a dose-dependent manner for 48 hrs. By a light microscope, CHO cells co-cultured with N. fowleri trophozoites (group I) for 48 hrs showed severe morphological destruction. On the contrary, CHO cells co-cultured with N. fowleri trophozoites and anti-Nfa1 polyclonal antibody (1:100 dilution) (group II) showed less destruction. In the LDH release assay results, group I showed 50.6% cytotoxicity, and group II showed 39.3%. Consequently, addition of an anti-Nfa1 polyclonal antibody produced a decreasing effect of in vitro cytotoxicity of N. fowleri in a dose-dependent manner.     

Biological factors affecting enflagellation of Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate that can be evoked to transform from amoebae to flagellates by subculture to nonnutrient buffer. More than half of the amoebae of strains KUL, nN68, and Lovell became enflagellated 300 min after subculture to amoeba-saline, whereas no amoebae of strains NF66, NF69, and HB4 did. N. fowleri nN68 enflagellated best when grown at 32 or 37 degrees C and subcultured to amoeba-saline at 37 or 42 degrees C. Amoebae from the stationary phase of growth enflagellated more readily than did actively growing amoebae. Incubation in expended culture medium from stationary-phase cultures enhanced the capability of growing amoebae to enflagellate after subculture to amoebasaline. Enflagellation was more extensive when the population density in amoebasaline did not exceed 2 x 10(5) amoebae per ml. Cycloheximide at 1 microgram/ml and actinomycin D at 25 micrograms/ml inhibited growth of N. fowleri nN68. Cycloheximide at 0.5 microgram/ml and actinomycin D at 25 micrograms/ml completely prevented enflagellation when added at time zero. Cycloheximide at 0.5 microgram/ml, added 120 to 300 min after initiation of enflagellation, prevented further differentiation and caused existing flagellates to revert to amoeboid cells. Similarly, actinomycin D at 25 micrograms/ml, added 90 to 300 min after initiation of enflagellation, retarded differentiation and caused flagellates to revert. Radiolabeled precursors were incorporated into macromolecules during differentiation in nonnutrient buffer. Enflagellation of N. fowleri is a suitable model for studying regulation of a eucaryotic protist.     

Computational identification of putative miRNAs and their target genes in pathogenic amoeba Naegleria fowleri

Naegleria fowleri is a parasitic unicellular free living eukaryotic amoeba. The parasite spreads through contaminated water andcauses primary amoebic meningoencephalitis (PAM). Therefore, it is of interest to understand its molecular pathogenesis. Hence,we analyzed the parasite genome for miRNAs (microRNAs) that are non-coding, single stranded RNA molecules. We identified245 miRNAs using computational methods in N. fowleri, of which five miRNAs are conserved. The predicted miRNA targets wereanalyzed by using miRanda (software) and further studied the functions by subsequently annotating using AmiGo (a geneontology web tool).     

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.     

Biochemical and functional characterization of a membrane-associated pore-forming protein from the pathogenic ameboflagellate Naegleria fowleri

A membrane-bound cytolytic pore-forming protein (N-PFP) produced by the pathogenic ameboflagellate Naegleria fowleri was characterized. N-PFP was solubilized from ameba membranes by detergent and enriched 300-fold by gel filtration chromatography. When analyzed by gel electrophoresis, N-PFP migrates with a molecular mass of 66 kDa and 50-54 kDa, under reducing and non-reducing conditions, respectively. In addition to lysing erythrocytes, N-PFP is cytotoxic to several tumor cell lines tested. Its hemolytic activity is not dependent on the presence of divalent cations. N-PFP rapidly depolarizes the membrane potential of microelectrode-impaled chicken embryo myocytes, suggesting that functional channel formation may represent the mode of membrane damage. In planar bilayers, N-PFP forms ion channels with heterogeneous unit conductances ranging between 150 and 400 picosiemens in 0.1 M NaCl and that are relatively resistant to closing by high voltages. Upon heat treatment (75 degrees C, 30 min), N-PFP forms channels with unit conductances that are on average larger than those formed by untreated N-PFP. N-PFP channels are slightly more permeable to cations than to anions. Using a liposome swelling-shrinkage assay, the functional diameter of N-PFP channels is estimated to range between 3.6 and 5.2 nm. N-PFP is immunologically distinct from the PFP/perforin produced by lymphocytes, the terminal components of complement and a PFP from the ameba Entamoeba histolytica, all of which produce pores on target membranes. This protein may have a direct lytic role during target cell killing mediated by N. fowleri.     

Geographic origin and spread of pathogenic Naegleria fowleri deduced from restriction enzyme patterns of repeated DNA

The restriction enzyme patterns of repeated DNA from 20 Naegleria fowleri and 13 N. gruberi strains were compared. On this basis strains of N. fowleri could be easily separated from N. gruberi. Although the restriction enzyme profiles of N. fowleri strains are quite homogenous, strains originating from Europe and from Australia had slightly different patterns. Both European and Australian profiles were found in the USA. In Australia a genetic variant was detected that is the same as the N. fowleri type present in New Zealand. Profiles of strains from India were identical with those from Europe. These results give additional information on the probable origin and dispersal of N. fowleri in the world. Strains of N. gruberi exhibit much more heterogenous banding patterns. Four European N. gruberi strains were, however, nearly identical while two strains from New Zealand had only a single band difference with one restriction enzyme. One Australian strain of N. gruberi was identical to an American strain that has been in culture for almost 30 years. The presence of a virus-like particle in a sister strain of this American isolate did not affect its banding patterns. Other strains of N. gruberi from the USA had diverse restriction enzyme patterns. Adelphamoeba galeacystis has restriction enzyme profiles distinct from those of the Naegleria strains investigated. Isoenzyme analysis in agarose isoelectric focusing confirmed the existence of intraspecific differences in N. fowleri.     

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.