RESUMO
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.
Assuntos
Interações Hospedeiro-Parasita/imunologia , Naegleria fowleri/metabolismo , Neutrófilos/fisiologia , Oxirredutases/biossíntese , Peroxidase/fisiologia , Proteínas de Protozoários/biossíntese , Compostos de Anilina/farmacologia , Animais , Forma Celular , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/ultraestrutura , Indução Enzimática , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Naegleria fowleri/enzimologia , Naegleria fowleri/crescimento & desenvolvimento , Naegleria fowleri/ultraestrutura , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Oxirredução , Estresse Oxidativo , Oxirredutases/genética , Peroxidase/antagonistas & inibidores , Proteínas de Protozoários/genética , Espécies Reativas de Oxigênio , Superóxidos/metabolismo , Vacúolos/ultraestruturaRESUMO
AIM: The aim of this work was to identify, characterize and evaluate the pathogenic role of mucinolytic activity released by Naegleria fowleri. MATERIALS & METHODS: Zymograms, protease inhibitors, anion exchange chromatography, MALDI-TOF-MS, enzymatic assays, Western blot, and confocal microscopy were used to identify and characterize a secreted mucinase; inhibition assays using antibodies, dot-blots and mouse survival tests were used to evaluate the mucinase as a virulence factor. RESULTS: A 94-kDa protein with mucinolytic activity was inducible and abolished by p-hydroxymercuribenzoate. MALDI-TOF-MS identified a glycoside hydrolase. Specific antibodies against N. fowleri-glycoside hydrolase inhibit cellular damage and MUC5AC degradation, and delay mouse mortality. CONCLUSION: Our findings suggest that secretory products from N. fowleri play an important role in mucus degradation during the invasion process.
Assuntos
Glicosídeo Hidrolases/metabolismo , Mucinas/metabolismo , Naegleria fowleri/enzimologia , Fatores de Virulência/metabolismo , Animais , Western Blotting , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/efeitos dos fármacos , Humanos , Hidroximercuribenzoatos/farmacologia , Camundongos , Microscopia Confocal , Naegleria fowleri/efeitos dos fármacos , Naegleria fowleri/metabolismo , Naegleria fowleri/patogenicidade , Polissacarídeo-Liases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.
Assuntos
Infecções Protozoárias do Sistema Nervoso Central/metabolismo , Cisteína Proteases/metabolismo , Interações Hospedeiro-Patógeno , Ferro/metabolismo , Proteólise , Animais , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/patologia , Proteínas de Ligação ao Ferro/metabolismo , Lactoferrina/metabolismo , Naegleria fowleri/enzimologia , Naegleria fowleri/patogenicidade , Bulbo Olfatório/metabolismo , Bulbo Olfatório/patologia , Transferrina/metabolismo , Trofozoítos/metabolismoRESUMO
Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Naegleria fowleri/enzimologia , Naegleria fowleri/crescimento & desenvolvimento , Fosfopiruvato Hidratase/metabolismo , Proteínas de Protozoários/metabolismo , Naegleria fowleri/genética , Fosfopiruvato Hidratase/genética , Proteínas de Protozoários/genéticaRESUMO
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.
Assuntos
Naegleria/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Western Blotting , Catálise/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Eletroforese em Gel de Poliacrilamida , Gelatina/metabolismo , Naegleria fowleri/enzimologia , Inibidores de Proteases/farmacologia , Prótons , Especificidade por SubstratoRESUMO
This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.
Assuntos
Acanthamoeba/efeitos dos fármacos , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antipsicóticos/farmacologia , NADH NADPH Oxirredutases/efeitos dos fármacos , Naegleria fowleri/efeitos dos fármacos , Acanthamoeba/enzimologia , Animais , Cromatografia Líquida de Alta Pressão , Glutationa Redutase/efeitos dos fármacos , Glutationa Redutase/metabolismo , Humanos , NADH NADPH Oxirredutases/metabolismo , Naegleria fowleri/enzimologiaRESUMO
Five Naegleria strains isolated from patients with primary amebic meningoencephalitis and one strain isolated from the water of an artificial canal were investigated. All strains were pathogenic for mice when instilled intranasally and showed cytopathic effects in Vero cell cultures. Their growth characteristics (isolation and subculture at 45 degrees C), serological results, and isoenzyme patterns permitted us to identify the six strains as Naegleria fowleri. This is the first time that Naegleria fowleri has been isolated from patients with primary amebic meningoencephalitis in Mexico.