Natural killer cell activity in Naegleria fowleri infected mice]

The natural killer (NK) cell activity of splenocytes and recycling capacity of NK cells were observed by combining the 51Cr-release cytotoxicity assay and single cell cytotoxicity assay against YAC-1. The ICR mice were infected intranasally with Naegleria fowleri, that is a pathogenic free-living amoeba. The mice infected with 1 x 10(5) trophozoites showed mortality rate of 76.7% and mean survival time of 12.9 days. The cytotoxic activity of NK cells in infected mice was significantly higher than that of non-infected mice during the period between 12 hours and day 3 after infection, and highest on day 1. The target-binding capacity of NK cells in infected mice was not different from that of non-infected ones. Maximal killing potential and maximal recycling capacity were remarkably increased in infected mice as compared with the control. The results obtained in this observation indicated that elevated NK cell activity in mice infected with N. fowleri was not due to target-binding capacity of NK cells but due to the increased activity of NK cells and increased recycling capacity of individual NK cells.     

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.     

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.     

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.     

Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I

In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.     

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.     

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.     

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.     

Susceptibility of wild mammals to infection with Naegleria fowleri

Animals of 4 families of small wild mammals were live-trapped and inoculated intranasally with Naegleria fowleri to determine patterns of susceptibility. Of the 7 species of animals examined, only rodents were susceptible to N. fowleri. Susceptible animals were eastern gray squirrel, hispid cotton rat, muskrat, and house mouse. Mammals that were not susceptible at a dose of 10(6) were opossum, raccoon, and eastern cottontail rabbit. Perhaps rodents and humans share a common anatomical or physiological determinant that makes them susceptible to infection with N. fowleri.     

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.     

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.     

Genetic Variation in the Free-Living Amoeba Naegleria fowleri

In this study, 30 strains of the pathogenic free-living amoeba Naegleria fowleri were investigated by using the randomly amplified polymorphic DNA (RAPD) method. The present study confirmed our previous finding that RAPD variation is not correlated with geographical origin. In particular, Mexican strains belong to the variant previously detected in Asia, Europe, and the United States. In France, surprisingly, strains from Cattenom gave RAPD patterns identical to those of the Japanese strains. In addition, all of these strains, together with an additional French strain from Chooz, exhibited similarities to South Pacific strains. The results also confirmed the presence of numerous variants in Europe, whereas only two variants were detected in the United States. The two variants found in the United States were different from the South Pacific variants. These findings do not support the previous hypothesis concerning the origin and modes of dispersal of N. fowleri.     

Occurrence and pathogenicity of Naegleria fowleri in artificially heated waters.

The occurrence of pathogenic Naegleria fowleri in thermal discharges, recipient waters, and cooling towers of eight power plants located in western Pennsylvania was investigated for 2 years in conjunction with several environmental measurements. Pathogenic N. fowleri was detected in one cooling tower and in the discharge, receiving waters, or both of five of eight localities. The occurrence of this organism was related to elevated temperatures, but no significant correlation was found for other biological and chemical parameters. Laboratory experiments on the effect of pH on pathogenic N. fowleri documented 100% survival at a range from 2.1 to 8.15. Higher pH reduced or killed the amoebae. No case of human primary amoebic meningoencephalitis occurred during the study.     

Calcium-dependent protection from complement lysis in Naegleria fowleri amebae

Pathogenic Naegleria fowleri amebae are resistant to the lytic effects of serum complement. The presence of surface glycoproteins or removal of the membrane attack complex (MAC) of complement from the cell surface by vesiculation serve to protect the amebae from complement lysis. The specific mediators important in stimulating complement resistance are not defined. These studies were undertaken to examine the effect of Ca(2+) ions in initiating complement resistance of N. fowleri in contrast to non-pathogenic complement-sensitive N. gruberi. Chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) or chelation of intracellular calcium with 1,2-bis-(O-Aminophenoxy) ethane-N,N,N,N tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) increased complement lysis of N. fowleri. Chelation of calcium ions did not affect complement sensitivity of N. gruberi. Increased lysis of ionomycin-treated N. fowleri was detected after exposure to serum complement, suggesting that a threshold level of Ca(2+) mediates complement resistance before survival mechanisms are overwhelmed and lysis occurs. A differential influx of Ca(2+) ions occurred in fura-2 labeled N. fowleri after deposition of complement component C9 to form the MAC complex on the cell surface in comparison to N. gruberi. These studies suggest that Ca(2+) ions influence complement resistance in N. fowleri but do not play a role in altering the sensitivity of N. gruberi to complement.