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.     

Amebostomes of Naegleria fowleri1

The strain of ameba, culture incubation temperature, and phase of ameba growth affected the number of amebostomes present on amebae of Naegleria fowleri. Serial passage of N. fowleri through mice decreased the average number of amebostomes. Amebostomes were shown to be functional by their ability to engulf yeast cells.     

Digestive proteolysis organization in two closely related Tenebrionid beetles: red flour beetle (Tribolium castaneum) and confused flour beetle (Tribolium confusum)

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6–6.0 in the anterior to 7.0–7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p‐nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin‐, chymotrypsin‐, and elastase‐like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases. © 2009 Wiley Periodicals, Inc.     

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.     

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.     

Ultrastructural Observations of Experimental Naegleria Meningoencephalitis in Mice: Intranuclear Inclusions in Amebae and Host Cells*

SYNOPSIS. Primary amebic meningoencephalitis was experimentally produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality, with average time of onset of symptoms or death occurring on the 7–8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.     

Antiparasitic Activity of Auranofin against Pathogenic Naegleria fowleri

We report that the gold containing antirheumatoid drug auranofin is amoebicidal against human pathogenic Naegleria fowleri. Treatment of N. fowleri cultures at biologically relevant concentrations of 0.75–3.0 μg/ml auranofin reduced amoeba counts, metabolic activity, and increased cell permeability. These results suggest that the addition of auranofin may benefit the treatment of N. fowleri‐infected patients afflicted by the rapidly fatal disease primary amoebic meningoencephalitis.     

Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment

We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40 kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38 kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40 kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40 kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.     

Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.     

A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.     

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.     

Conditions for Maximum Enflagellation in Naegleria fowleri1

ABSTRACT. Ameba to flagellate transformation in Naegleria fowleri (Lovell strain) was affected by growth temperature, phase of growth, strain of ameba, culture agitation, enflagellation temperature, enflagellation diluent, and cell concentration. Amebae transformed best when they were grown without agitation and enflagellated with agitation. Regardless of growth temperature (23°, 30°, 37°, and 42°C were tested), amebae transformed best at 37°C. Enflagellation was greatest for cells harvested between 24 h (mid-exponential) and 84 h (late stationary) of growth.     

Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

Aims: The potential effect of in‐premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free‐living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Methods and Results: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington‐2 increased in growth at 30, 32 and 37°C while strains Philadelphia‐1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington‐2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia‐1 in the presence of N. fowleri. Yet, strain Bloomington‐2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human‐pathogenic legionellae and point to future research required to better understand Legionella ecology.     

Detection of Biomarkers of Pathogenic Naegleria fowleri Through Mass Spectrometry and Proteomics

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix‐assisted laser‐desorption‐ionization‐time‐of‐flight mass spectrometry MALDI‐TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF‐TOF instrument. MALDI‐TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI‐TOF MS fingerprinting is a rapid, reproducible, high‐throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.     

Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.     

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.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

Attachment and Short-Term Maintenance of Motility and Viability of Entamoeba Histolytica In A Defined Medium*

Organic requirements for attachment to glass, elongation, and motility of Entamoeba histolytica, have been determined. the trophozoite, which has been grown axenically only in highly complex media with reduced oxygen tensions, remains rounded and detached when placed in a Tris-HCl buffered solution containing NaCI, KCI, MgCI₂, and CaCI₂. A maintenance medium in which the amebae could attach to glass, elongate, and remain motile and viable for 12 to 24 h was devised with the addition of cysteine, ascorbic acid, bovine serum albumin, and the vitamin solution of medium NCTC #107. Tris-HCI was the most effective buffer tested and the optimal pH was 6.9 to 7.0. Survival, but not attachment, of the amebae was decreased at osmolalities ranging between 110 and 180 milliosmoles/kg, whereas both functions were decreased above ～260 milliosmoles/kg. Bovine serum albumin, the most effective of the proteins tested, and the vitamin solution helped maintain attachment of some ameba strains, but were not required by other strains. the requirements for cysteine and ascorbic acid were absolute and highly specific. During incubation in the maintenance medium, cell volumes decreased. Sensitivity of the organisms to agglutination by concanavalin A, wheat germ agglutinin, soybean agglutinin and fucose binding protein remained unchanged.     

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.     

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC

Abstract A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-β-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50 909 Da. The enzyme is functional and extremely overexpressed in E. coli .