Phagocytic activity of three Naegleria strains in the presence of erythrocytes of various types

The phagocytic activities of N. lovaniensis (Aq/9/1/45D) and N. gruberi (1518/1f and 1518/1e) were studied in the presence of erythrocytes of various species: chicken, rabbit, goat, and human (A+, B+, and AB+ were tested). The percentage of amoebae with ingested red cells, the phagocytic index (PhI), can be considered as an expression of phagocytic activity. Under given conditions (erythrocyte concentration, incubation time, age of amoebic cultures) each strain of Naegleria prefers one erythrocyte type. Thus, for 72-h cultures, N. lovaniensis ingested more A+ type erythrocytes than did N. gruberi strains but had very low affinity for rabbit red cells except when very high concentrations were tested. Naegleria gruberi 1f was the most active of the three strains towards rabbit and B+ and AB+ human erythrocytes, but very low PhIs were obtained with goat erythrocytes. Naegleria gruberi 1e exhibited high phagocytic activity for every erythrocyte type except for rabbit red cells.     

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.     

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.     

Quantitative Detection and Differentiation of Free-Living Amoeba Species Using SYBR Green–Based Real-Time PCR Melting Curve Analysis

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.     

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.     

Phagocytosis of erythrocytes by Acanthamoeba sp

Phagocytic recognition by the unicellular soil organism Acanthamoeba sp. (Neff strain) was examined with fresh or modified erythrocytes. Several parameters were studied of the interaction of glutaraldehyde-treated red cells with amoebae attached to glass. Attachment and ingestion steps of particle uptake were found to have differing temperature dependence. Particle-phagocyte interaction required the addition of Na⁺ or Ca²⁺ and was inhibited by high osmolarity or ionic strength. These features are similar to those previously described for mammalian macrophages. A quantitative spectrophotometric technique was adapted to the measurement of erythrocyte uptake after lysis of noningested red cells. Rates of uptake of six species of red cells spanned a 100-fold range. While untreated sheep red cells were taken up at very low rates, ingestion of red cells treated with aldehyde, tannic acid, polylysine, carbodiimide, ferrous sulfate or salt-free sucrose was appreciably increased. Some but not all of these modified red cells were previously found to interact with macrophages and insect hemocytes. Thus Acanthamoeba displays phagocytic recognition of untreated and modified erythrocytes. The results also indicate that the particle vocabulary ingested by the amoebae overlaps in part with that of certain metazoan phagocytes.     

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.     

Ultrastructure of Cysts of Naegleria spp: A Comparative Study*

SYNOPSIS. Ultrastructure of cysts of Naegleria gruberi, Naegleria fowleri, and Naegleria jadini was compared by transmission electron microscopy. Pores in the cyst wall were observed in all 3 species. In N. gruberi they were surrounded by a collar and sealed with a relatively large mucoid plug; no such collar was seen around the pores in the other 2 species, in which the plug was smaller than that in N. gruberi. An electron-dense plaque serving as an additional pore closure was present in all 3 species. In N. gruberi, the cyst wall consisted of an inner thick and an outer thin layer; however, only the inner component was present in cysts of N. fowleri and N. jadini, which had a smooth appearance. At the ultrastructural level, excystment of N. fowleri involved digestion of the mucoid plug and emergence of the trophozoite through the pore. Some digestion of the cyst wall also appeared to take place during excystment.     

Target cell deformability determines the type of phagocytic mechanism used by Entamoeba histolytica-like,Laredo strain

Summary— Morphological study of red blood cell phagocytosis by Entamoeba histolytica-like (Laredo strain) has shown that this amoeba is able to ingest by two distinct mechanisms. One is classical phagocytosis and the other is by suction or microphagocytosis. Rigidification of red blood cells by treatment with glutaraldehyde shows that there is a correlation between the deformability of the ingested cell and the type of phagocytosis observed. Indeed, as the red cells become more rigid, less microphagocytosis is observed. To demonstrate that this shift in phagocytic mechanisms is not induced by the modification of a surface receptor by the glutaraldehyde treatment, the amoebas were fed with erythrocyte ghosts. Since these have lost most of their hemoglobin content, they are less rigid than the intact erythrocytes. The ghosts, even after glutaraldehyde treatment, are always ingested by microphagocytosis. These results have therefore led us to conclude that the type of erythrocyte phagocytosis used by E histolytica-like (Laredo strain) is determined by the deformability of the targetted red blood cells.     

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.     

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.     

A comparative study of the membrane proteins from Naegleria species: A 23-kDa protein participates in the virulence of Naegleria fowleri

The plasma membrane is essential in the pathogenicity of several microorganisms. However, to date, there are few studies related to the plasma membrane proteins in Naegleria fowleri; this amoeba produces a fatal disease called primary amoebic meningoencephalitis. In the present study, we analyzed the electrophoretic pattern of the membrane proteins of N. fowleri and compared it with the nonpathogenic N. lovaniensis and N. gruberi. We detected a 23-kDa protein (Nf23) present at a higher level in N. fowleri than in the nonpathogenic amoebae. The mass spectrometry analysis showed that the Nf23 protein has a sequence of 229 amino acids that corresponds to a membrane protein. The mRNA level of nf23 was overexpressed 4-fold and 40,000-fold in N. fowleri compared with N. lovaniensis and N. gruberi, respectively. Moreover, we found a 5-fold overexpression of nf23 in N. fowleri trophozoites recovered from mouse brains compared with trophozoites axenically cultivated. In addition, the cytopathic effect on Madin-Darby Canine Kidney cells coincubated with N. fowleri diminished in the presence of antibodies against Nf23; nevertheless, the nonpathogenic amoebae did not produce damage to the monolayer cells. These results suggest that the plasma membrane protein Nf23 is probably involved in the virulence of N. fowleri.     

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.     

Preparation and Properties of Mitochondria from Naegleria gruberi*

Whole cell respiration rates were measured polarographically for Naegleria gruberi during growth in agitated cultures. Log growth phase amebae consumed 80 ng atoms O/min/mg cell protein. At stationary phase, respiration rate decreased 4–fold. Intact mitochondria were isolated from N. gruberi and their oxidative and phosphorylative capacities were studied polarographically. As with the mammalian system, the mitochondria oxidized succinate and NAD-linked substrates, but unlike rat liver mitochondria, those from the protozoan rapidly oxidized citrate and NADH. The rates of substrate oxidation were ADP-dependent, with ADP:O ratios equalling ? 2.8 for NAD-linked substrates and ? 2.2 for succinate. The respiratory control ratios. 2 to 4 for 11 substrates, were dependent on Pi, Mg²⁺, and serum albumin. Potassium cyanide, azide, malonale, and rotenone inhibited electron transport the same way as that of the mammalian system: however, amytal inhibited both glutamate and succinate respiration. Pentachlorophenol, DNP, and bilirubin uncoupled oxidation from phosphorylation. Difference spectra of oxidized and dithionite-reduced mitochondria had distinct absorption bands of flavins and of c-, b-, and α-type cytochromes.     

Studies on the genetic polymorphism of lactate dehydrogenase B (phenotype B−) in rodent erythrocytes

While the common red cell phenotype of lactate dehydrogenase in mammals includes A and B subunits, in some species, especially of the rodent suborder Myomorpha, LDH B subunits are absent in erythrocytes (phenotype B^–). A polymorphism involving LDH B⁺ and LDH B^– phenotypes was observed in Mus musculus, and it was found that LDH B^– is recessive in relation to LDH B⁺ (Shows and Ruddle, 1968). A similar polymorphism is described here in the field mouse, Apodemus sylvaticus. In family studies, the recessive mode of inheritance is confirmed. Induction of reticulocytosis reveals that the LDH B^– phenotype is not the consequence of degradation of the B subunits in aging red cells. The nature of the mutation giving rise to this polymorphism is discussed.Supported by the Deutsche Forschungsgemeinschaft.     

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.     

Ameboflagellate transformation I. Possible initiation of the ameba-to-flagellate transformation of Naegleria gruberi (NB-1) by divalent cation influx

The hypothesis that the ameba-to-flagellate transformation (AFT) of Naegleria gruberi (NB-1) requires Ca²⁺ and/or Mg²⁺ was tested with divalent cation blockers and competitors. Several compounds were found to decrease the rate and magnitude of the AFT as well as increasing the time of enflagellation onset. The compounds include: the chloride salts of lanthanum, cobalt, and manganese; C-600; and EDTA. These results suggest that the AFT requires the influx of Ca²⁺ and/or Mg²⁺.Supported by NIH Training Grant 5-T01-N505670     

Iron economy in Naegleria gruberi reflects its metabolic flexibility

Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process.     

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.     

Homeostasis of Sodium and Potassium Ions in Erythrocytes of the Lamprey Lampetra fluviatilis: Effect of Ion Transport and Metabolic Blockers, and Ionophores

After incubation of lamprey Lampetra fluviatilis erythrocytes in the standard medium for 90–120 min, intracellular Na⁺ and K⁺ content remained unchanged (28.7 ± 1.1 and 66.3 ± 1.5 mmol/l cells, respectively, n = 33). The erythrocyte ion content also did not change after treatment of the cells with ion transport inhibitors, Ba^{2 +} and amiloride. Addition of 0.1 mM ouabain to the incubation medium led to a decrease of K⁺ content by 8.4 ± 1.2 and to an increase of Na⁺ content by 2.4 ± 0.8 mmol/l/2 h. Similar reciprocal changes in the cellular ion composition were observed after treatment of the erythrocytes by oxidative metabolism inhibitors (rotenone and CCCP—carbonyl cyanide m-chlorophenyl-hydrazone). The metabolic blockers produced more significant ion composition changes in comparison with ouabain. An increase of intracellular Na⁺ content under effect of CCCP was completely inhibited by amiloride. It can be suggested that inhibition of oxidative metabolism is accompanied by a cell acidification and Na⁺/H⁺ exchange activation. Erythrocyte acidification by a K⁺/H⁺ ionophore led to a rapid cellular Na⁺ accumulation, which indicates the presence of a Na⁺/H⁺ exchanger with high activity. The K⁺ ionophore valinomycin produced a relatively small K⁺ loss from the lamprey erythrocytes to indicate a low anion conductance of the cells. The data obtained indicate an important role of oxidative metabolism in the monovalent ion homeostasis in the lamprey red blood cells.