The MPO system participates actively in the formation of an oxidative environment produced by neutrophils and activates the antioxidant mechanism of Naegleria fowleri.

Naegleria fowleri produces a fatal disease called primary amebic meningoencephalitis (PAM), which is characterized by an extensive inflammatory reaction in the CNS. It is known that the immune response is orchestrated mainly by neutrophils, which activate several defense mechanisms in the host, including phagocytosis, the release of different enzymes such as myeloperoxidase (MPO), and the production of neutrophil extracellular traps. However, the mechanisms by which amoebas evade the neutrophil response are still unknown. In this study, we analyzed the ability of N. fowleri to respond to the stress exerted by MPO. Interestingly, after the interaction of trophozoites with neutrophils, the amoeba viability was not altered; however, ultrastructural changes were observed. To analyze the influence of MPO against N. fowleri and its participation in free radical production, we evaluated its enzymatic activity, expression, and localization with and without the specific 4-aminobenzoic acid hydrazide inhibitor. The production of oxidizing molecules is the principal mechanism used by neutrophils to eliminate pathogens. In this context, we demonstrated an increase in the production of NO, superoxide anion, and reactive oxygen species; in addition, the overexpression of several antioxidant enzymes present in the trophozoites was quantified. The findings strongly suggest that N. fowleri possesses antioxidant machinery that is activated in response to an oxidative environment, allowing it to evade the neutrophil-mediated immune response, which may contribute to the establishment of PAM.

CYP51 is an essential drug target for the treatment of primary amoebic meningoencephalitis (PAM).

Primary Amoebic Meningoencephalitis (PAM) is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered "rare" (but likely underreported) the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug 'repurposing'-a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51) target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i) systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii) identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii) analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially 'druggable' target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.

Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds and is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). PAM occurs when amoebae attach to the nasal epithelium and invade the CNS, a process that involves binding to, and degradation of, extracellular matrix (ECM) components. This degradation is mediated by matrix metalloproteinases (MMPs), enzymes that have been described in other pathogenic protozoa, and that have been linked to their increased motility and invasive capability. These enzymes also are upregulated in tumorigenic cells and have been implicated in metastasis of certain tumours. In the present study, in vitro experiments linked MMPs functionally to the degradation of the ECM. Gelatin zymography demonstrated enzyme activity in N. fowleri whole cell lysates, conditioned media and media collected from invasion assays. Western immunoblotting indicated the presence of the metalloproteinases MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 [membrane type-1 matrix metalloproteinase (MT1-MMP)]. Highly virulent mouse-passaged amoebae expressed higher levels of MMPs than weakly virulent axenically grown amoebae. The functional relevance of MMPs in media was indicated through the use of the MMP inhibitor, 1,10-phenanthroline. The collective in vitro results suggest that MMPs play a critical role in vivo in invasion of the CNS and that these enzymes may be amenable targets for limiting PAM.

ANTI-AMEBIC ACTIVITY OF DIOSGENIN ON NAEGLERIA FOWLERI TROPHOZOITES.

The aim of this study was to investigate the activity of diosgenin against Naegleria fowleri trophozoites at the cellular and molecular levels. Diosgenin (100 µg/ml; 241.2 µM) had a 100% inhibitory effect on N. fowleri trophozoites (5 x 10(5) cell/ml). Scanning electron micrograph revealed diosgenin decreased the number of sucker-like apparatuses and food cup formation among N. fowleri trophozoites at 3 and 6 hours post-exposure, respectively. Diosgenin down-regulated the nf cysteine protease gene expression of N. fowleri trophozoites at 6 and 12 hours post-exposure. The toxicity to mammalian cells caused by diosgenin at therapeutic dose was less than amphotericin B, the current drug used to treat N. fowleri infections. Our findings suggest diosgenin has activity against the surface membrane and the nf cysteine pro tease of N. fowleri trophozoites. However, the other mechanisms of action of diosgenin against N. fowleri trophozoites require further exploration.

Naegleria fowleri: contact-dependent secretion of electrondense granules (EDG).

The free living amoeba Naegleria fowleri is pathogenic to humans but also to other mammalians. These amoebae may invade the nasal mucosa and migrate into the brain causing cerebral hemorrhagic necrosis, a rapidly fatal infection. Knowledge of the cytolytic mechanism involved in the destruction of brain tissues by Naegleria trophozoites is limited. In other amoebic species, such as Entamoeba histolytica, we have previously reported the possible lytic role of small cytoplasmic components endowed with proteolytic activities, known as electrondense granules (EDG). Using transmission electron microscopy we now report that EDG, seldom found in long term cultured N. fowleri, are present in abundance in trophozoites recovered from experimental mice brain lesions. Numerous EDG were also observed in amoebae incubated with collagen substrates or cultured epithelial cells. SDS-PAGE assays of concentrated supernatants of these trophozoites, containing EDG, revealed proteolytic activities. These results suggest that EDG may have a clear role in the cytopathic mechanisms of this pathogenic amoeba.

Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

Scanning electron microscopic study of trophozoite and cyst stages of Naegleria fowleri.

Whole trophozoites and cysts of axenically cultivated Naegleria fowleri were prepared for study of their surface morphology by scanning electron microscopy (SEM). Trophozoites and cyst stages were studied from Chang's culture media. Some trophozoites were examined after animal inoculation and brain isolation to compare the changes in surface features. Photomicrographs of freeze-dried and critical point-dried organisms fixed with glutaraldehyde were presented along with views of both isolates of trophozoites to compare the surface features. SEM revealed the surface of trophozoites to be undulating, wrinkled and covered at irregular intervals by protruding vesicles. There were also surface extensions which were long and thin in brain isolates which may help in the contact and cytolysis of host cells at some distance from the trophozoite. Some cysts appeared wrinkled while others smooth, and empty cysts were also seen with many pores on the surface.

Naegleria fowleri: light and electron microscopy study of mitosis.

DAPI and Feulgen stains were used as specific DNA markers for studying the mitosis process in Naegleria fowleri. Both DAPI and Feulgen stains reacted with DNA in the nuclei of the amoebae. Representative figures of N. fowleri mitotic nuclei with a defined arrangement according to the phase of the cell cycle were observed. A notable characteristic is that the nucleolus is present throughout the stages of mitosis. During metaphase, several deeply stained DNA condensations following an elongated pattern were observed, corresponding almost certainly to tightly grouped chromosomes. Ultrastructural observations demonstrated that the nucleus divides by cryptomitosis, a process in which the nuclear membrane does not disappear during the mitosis. Centrioles were not found, and a spindle of microtubules was observed running the length of the nucleus from pole to pole however, they did not come to a focal point.

[Amebic meningoencephalitis].

It has been reported that amebic meningoencephalitis is caused by some rhizopods, which are taxonomically different from Entamoeba histolytica which is well known as the causative agent of amebic dysentery. Different types of human meningoencephalitis have been reported to be caused by amphizoic amebae, which are not obligatorily parasitic (endozoic) but are usually free-living (exozoic) in nature, i.e., in environmental water and soil: Naegleria fowleri causes acute primary amebic meningoencephalitis (PAM). Acanthamoeba spp. and Balamuthia mandrillaris produce chronic and opportunistic granulomatous amebic (meningo) encephalitis (GAE). Further, most recently, Sappinia diploidea has been identified as an agent that causes comparatively acute type of encephalitis.

Survey for the presence of Naegleria fowleri amebae in lake water used to cool reactors at a nuclear power generating plant.

Water from Lake Anna in Virginia, a lake that is used to cool reactors at a nuclear power plant and for recreational activities, was assessed for the presence of Naegleria fowleri, an ameba that causes primary amebic meningoencephalitis (PAM). This survey was undertaken because it has been reported that thermally enriched water fosters the propagation of N. fowleri and, hence, increases the risk of infection to humans. Of 16 sites sampled during the summer of 2007, nine were found to be positive for N. fowleri by a nested polymerase chain reaction assay. However, total ameba counts, inclusive of N. fowleri, never exceeded 12/50 mL of lake water at any site. No correlation was obtained between the conductivity, dissolved oxygen, temperature, and pH of water and presence of N. fowleri. To date, cases of PAM have not been reported from this thermally enriched lake. It is postulated that predation by other protozoa and invertebrates, disturbance of the water surface from recreational boating activities, or the presence of bacterial or fungal toxins, maintain the number N. fowleri at a low level in Lake Anna.

Scanning electron microscopic study of human neuroblastoma cells affected with Naegleria fowleri Thai strains.

In order to understand the pathogenesis of Naegleria fowleri in primary amoebic meningoencephalitis, the human neuroblastoma (SK-N-MC) and African green monkey kidney (Vero) cells were studied in vitro. Amoeba suspension in cell-culture medium was added to the confluent monolayer of SK-N-MC and Vero cells. The cytopathic activity of N. fowleri trophozoites in co-culture system was elucidated by scanning electron microscope at 3, 6, 9, 12, and 24 h. Two strains of N. fowleri displayed well-organized vigorous pseudopods in Nelson's medium at 37 degrees C. In co-culture, the target monolayer cells were damaged by two mechanisms, phagocytosis by vigorous pseudopods and engulfment by sucker-like apparatus. N. fowleri trophozoites produced amoebostomes only in co-culture with SK-N-MC cells. In contrast, we could not find such apparatus in the co-culture with Vero cells. The complete destruction time (100%) at 1:1 amoeba/cells ratio of SK-N-MC cells (1 day) was shorter than the Vero cells (12 days). In conclusion, SK-N-MC cells were confirmed to be a target model for studying neuropathogenesis of primary amoebic meningoencephalitis.

Acanthamoeba castellanii: identification and distribution of actin cytoskeleton.

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparison between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.

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.

Modulation of a "CD59-like" protein in Naegleria fowleri amebae by bacteria.

Found in soil and freshwater habitats, Naegleria fowleri are free-living amebae that cause a fatal disease in humans called Primary Amebic Meningoencephalitis. In the natural environment, amebae feed on bacteria. In the infected host, the amebae lyse and ingest nerve tissue. Recently, we have established that N. fowleri expresses a "CD59-like" surface protein, but the function of this protein in the ameba has not been elucidated. In mammalian cells, CD59 is a complement-regulatory protein that inhibits complement-mediated lysis of cells expressing this protein. In the present study, expression of the "CD59-like" protein in response to bacteria and bacterial toxins was investigated by Western immunoblot analysis. Co-culture of N. fowleri with log phase Escherichia coli or Pseudomonas aeruginosa resulted in differential expression of the "CD59-like" protein. Co-cultures of amebae and bacteria were examined by electron microscopy. The results of our study implicate a possible protective role of the "CD59-like" protein in response to bacterial predators and bacterial toxins, because amebae remained intact after co-culture with bacteria.

Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri.

We previously cloned an antigenic gene (named nfa1) from a cDNA library of Naegleria fowleri by immunoscreening. The nfa1 gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of N. fowleri against Chinese hamster ovary (CHO) cells. Trophozoites of N. fowleri in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with N. fowleri, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. When examined using a light microscope, CHO cells grown in cocultures with N. fowleri trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with N. fowleri trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.

[Human pathology caused by free-living amoebae]. / Patologia umana da amebe a vita libera.

Naegleria fowleri, Acanthamoeba spp. and Balamuthia mandrillaris are free-living amoebae that occasionally may induce pathology in human beings. CNS disease due to N. fowleri, called "primary" amoebic meningoencephalitis, is acquired after exposure to polluted waters in swimming pools, rivers, and lakes. The clinical course is acute, often fulminant and characterized pathologically by necrotizing hemorrhagic meningoencephalitis, involving mainly the base of the brain, brainstem and cerebellum. In contrast, some Acanthamoeba spp. and B. mandrillaris cause opportunistic, chronic "granulomatous" encephalitis in subjects pathologically or iatrogenically immunocompromised. There are, most likely, foci of protozoa in lung and skin reaching the CNS by hematogenous route. Only Acanthamoeba spp. can also produce severe, subacute keratitis, mainly today in contact lens wearers.

Cocultivation of the amoeba Naegleria fowleri and the amoebicin- producing strain Bacillus licheniformis M-4.

Antagonism between Bacillus licheniformis M-4 and the pathogenic amoeba Naegleria fowleri HB-1 during cocultivation was influenced by the composition of the medium and the initial amoeba/bacterium ratio. While a ratio of 50 caused complete lysis of amoebae in soil extract with 0.3% glucose (SEG) before 72 h, this ratio had to be at least 12-fold lower in order to obtain similar results in Cline medium. Sporulation of B. licheniformis M-4 took place much earlier in SEG. Amoebicin production was stimulated by the presence of amoebae by either shortening the time of production (as in SEG) or increasing the amount of amoebicins released (as in Cline medium). Electron microscopy showed that amoebae cocultivated in the Cline medium contained bacteria enclosed in digestive vacuoles, while amoebae from SEG cocultures did not.

Membrane vesiculation of Naegleria fowleri amoebae as a mechanism for resisting complement damage.

Pathogenic and nonpathogenic Naegleria amoebae activate the alternative C pathway; however, only pathogenic amoebae are resistant to C-mediated damage. The present study was undertaken to determine the mechanism by which highly pathogenic N. fowleri amoebae resist C-mediated damage. Nomarski optics microscopy and electron microscopy of Naegleria amoebae revealed membrane blebbing on the surface of C-resistant N. fowleri, but not on C-sensitive N. gruberi, in response to incubation in normal human serum diluted 1:4 to 1:16. Immunofluorescent staining of pathogenic amoebae, by using antiserum to human C proteins comprising the membrane attack complex, C5b through C9, and FITC-labeled goat anti-rabbit IgG, confirmed that the membrane attack complex was concentrated on the membrane blebs. Binding studies with the use of radioiodinated C9 demonstrated a decrease in the 125I-labeled C9 cpm associated with N. fowleri amoebae and an increase in the 125I-labeled C9 cpm associated with the released membrane vesicles after increasing incubation periods in normal human serum. Treatment of pathogenic, C-resistant N. fowleri with cytochalasin D or cytochalasin B to inhibit actin-dependent exocytic processes increased the susceptibility of the amoebae to C damage. In contrast, incubation of nonpathogenic, C-sensitive amoebae with cytochalasins did not alter their susceptibility to C lysis. These data indicate that pathogenic N. fowleri use membrane vesiculation to remove membrane-deposited C proteins, specifically the membrane attack complex (C5b-C9). The ability to remove surface-associated membrane attack complexes serves as one mechanism by which pathogenic N. fowleri resist C lysis.

Modulation of biological functions of Naegleria fowleri amoebae by growth medium.

Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri, LEE, and a highly pathogenic strain, LEEmpC1, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri, LEE and LEEmpC1, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.