Ancient weapons for attack and defense: the pore-forming polypeptides of pathogenic enteric and free-living amoeboid protozoa

Pore-forming polypeptides have been purified from several amoeboid protozoans that are well-known human pathogens. Obligate enteric parasites, such as Entamoeba histolytica, and free-living but potentially highly pathogenic species, such as Naegleria fowleri, contain these cytolytic molecules inside cytoplasmic granules. Comprehensive functional and structural studies have been conducted that include isolation of the proteins from their natural sources, monitoring of their biological activity towards different targets, and molecular cloning of the genes of their precursors. In the case of the most prominent member of the protein family, with respect to protozoans, the three-dimensional structure of amoebapore A was solved recently. The amoebic pore-forming polypeptides can rapidly perforate human cells. The antibacterial activity of amoebapores and of related polypetides from free-living protozoa points to a more vital function of these molecules: inside the digestive vacuoles they combat growth of phagocytosed bacteria which are killed when their cytoplasmic membranes are permeabilized. The concommitant activity of these proteins towards host cells may be due to a coincidental selection for an efficient effector molecule. Nonetheless, several lines of evidence indicate that these factors are involved in pathogenesis of fatal diseases induced by amoeboid protozoa.     

The immune response to Naegleria fowleri amebae and pathogenesis of infection

The genus Naegleria is comprised of a group of free-living ameboflagellates found in diverse habitats worldwide. Over 30 species have been isolated from soil and water but only Naegleria fowleri (N. fowleri) has been associated with human disease. Naegleria fowleri causes primary amebic meningoencephalitis (PAM), a fatal disease of the central nervous system. The pathogenesis of PAM and the role of host immunity to N. fowleri are poorly understood. Strategies for combating infection are limited because disease progression is rapid and N. fowleri has developed strategies to evade the immune system. The medical significance of these free-living ameboflagellates should not be underestimated, not only because they are agents of human disease, but also because they can serve as reservoirs of pathogenic bacteria.     

Thermal ecology of Naegleria fowleri from a power plant cooling reservoir

The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis. N. fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined. In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp. before and after thermal additions from a nuclear power plant. Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp. and pathogenic N. fowleri. Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis. N. fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir. The probability of isolating thermophilic Naegleria and pathogenic N. fowleri increased significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.     

Thermal ecology of Naegleria fowleri from a power plant cooling reservoir.

The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis. N. fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined. In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp. before and after thermal additions from a nuclear power plant. Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp. and pathogenic N. fowleri. Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis. N. fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir. The probability of isolating thermophilic Naegleria and pathogenic N. fowleri increased significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.     

High-Resolution Polyacrylamide Gradient Gel Electrophoresis (PGGE) of Isoenzymes from Five Naegleria Species

ABSTRACT. High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis , two N. lovaniensis , one N. jadini , two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis, two N. lovaniensis, one N. jadini, two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

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.     

Cytolytic activity of Naegleria fowleri cell-free extract

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 degrees 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.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems

This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.     

Protein kinase activation and protein phosphorylation in Naegleria fowleri amebae in response to normal human serum

Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.     

Soluble amoebicidal factors mediate cytolysis of Naegleria fowleri by activated macrophages

Murine peritoneal macrophages activated in vivo with Corynebacterium parvum or bacille Calmette-Guérin, in contrast to resident macrophages, demonstrated significant cytolysis of the amoeba, Naegleria fowleri. Catalase and superoxide dismutase, both alone and in combination, failed to inhibit cytolysis of amoebae. N. fowleri amoebae demonstrated significant resistance to exogenously added hydrogen peroxide. The hydroxyl radical scavengers mannitol, thiourea, and dimethyl sulfoxide, as well as anaerobic conditions, failed to inhibit the amoebicidal activity of activated macrophages. Actinomycin D, cycloheximide, and puromycin blocked macrophage amoebicidal activity. Conditioned medium (CM) from lipopolysaccharide-stimulated, but not unstimulated, cultures of activated macrophages was capable of mediating cytolysis of N. fowleri amoebae. Cytolytic activity was recovered by ammonium sulfate precipitation of CM. Heat treatment of the CM inactivated cytolytic activity. Results indicate soluble proteins of activated macrophage origin to be responsible for the amoebicidal activity.     

Modification of low density lipoprotein metabolism by growth factors in cultured vascular cells and human skin fibroblasts. Dependence upon duration of exposure

Phospholipase A, sphingomyelinase and lysophospholipase activities were examined in cell homogenates and cell-free culture media of virulent and virulent-attenuated Naegleria fowleri and nonpathogenic Naegleria gruberi. Homogenates of virulent N. fowleri contained from 3 to 250 times the lipolytic activity of virulent-attenuated and non-pathogenic Naegleria spp. Similarly, the cell-free media of virulent N. fowleri cultures contained large quantities of phospholipase A, lysophospholipase and sphingomyelinase while comparable activities in the cell-free media of virulent-attenuated and nonpathogenic Naegleria spp. were only slightly, if at all, detectable. Lipolytic enzymes accumulated in the media of virulent N. fowleri cultures at various stages during growth but not in virulent-attenuated and nonpathogenic Naegleria cultures. In general, phospholipase A and sphingomyelinase accumulated during the log phase of growth while lysophospholipase appeared only in the late stationary phase. We conclude that pathogenic Naegleria contain potent lipolytic enzymes that are released selectively into the media during growth. These enzymes could contribute to the pathogenesis of Naegleria-induced primary amoebic meningoencephalitis.     

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

ABSTRACT: Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri , causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria , and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini , and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri , with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.     

Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake

Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.     

Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake.

Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.     

Antagonistic Action of the Bacterium Bacillus Licheniformis M-4 Toward the Amoeba Naegleria Fowleri

ABSTRACT. Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram-positive and Gram-negative bacteria. Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated. This substance (m-4) is stable at high temperature (up to 100°C) and extremes of pH (2.5-9.5) and also at -20°C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.     

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.     

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.