RESUMO
Naegleria fowleri, also known as the "brain-eating" amoeba, is a free-living protozoan that resides in freshwater bodies. This pathogenic amoeba infects humans as a casual event when swimming in contaminated water. Upon inhalation, N. fowleri invades the central nervous system and causes primary amoebic meningoencephalitis (PAM), a rapidly progressive and often fatal disease. Although PAM is considered rare, reducing its case fatality rate compels the search for pathogen-specific proteins with a structure-function relationship that favors their application as targets for discovering new or improved drugs against N. fowleri infections. Herein, we report a computational approach to study the structural features of Nf314 (a serine carboxypeptidase that is a virulence-related protein in N. fowleri infections) and assess its potential as a drug target, using bioinformatics tools and in silico molecular docking experiments. Our findings suggest that Nf314 has a ligand binding site suitable for the structure-based design of specific inhibitors. This study represents a further step toward postulating a reliable therapeutic target to treat PAM with drugs specifically aimed at blocking the pathogen proliferation by inhibiting protein function.
Assuntos
Infecções Protozoárias do Sistema Nervoso Central , Naegleria fowleri , Humanos , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Simulação de Acoplamento Molecular , Ligantes , Naegleria fowleri/metabolismo , Água/metabolismoRESUMO
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
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.
Assuntos
Naegleria fowleri/metabolismo , Naegleria fowleri/patogenicidade , Naegleria/metabolismo , Naegleria/patogenicidade , Proteínas de Protozoários/metabolismo , Virulência/genética , Animais , Encéfalo/metabolismo , Encéfalo/parasitologia , Cães , Expressão Gênica , Células Madin Darby de Rim Canino , Camundongos , Naegleria fowleri/genética , Proteínas de Protozoários/genética , Análise de Sequência de ProteínaRESUMO
The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleriafowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.
Assuntos
Acanthamoeba/patogenicidade , Entamoeba histolytica/patogenicidade , Células Epiteliais/parasitologia , Interações Hospedeiro-Parasita , Naegleria fowleri/patogenicidade , Infecções por Protozoários/parasitologia , Acanthamoeba/metabolismo , Animais , Entamoeba histolytica/metabolismo , Células Epiteliais/metabolismo , Humanos , Naegleria fowleri/metabolismo , Infecções por Protozoários/metabolismoRESUMO
Naegleria fowleri causes a fatal disease known as primary amoebic meningoencephalitis. This condition is characterized by an acute inflammation that originates from the free passage of peripheral blood cells to the central nervous system through the alteration of the blood-brain barrier. In this work, we established models of the infection in rats and in a primary culture of endothelial cells from rat brains with the aim of evaluating the activation and the alterations of these cells by N. fowleri. We proved that the rat develops the infection similar to the mouse model. We also found that amoebic cysteine proteases produced by the trophozoites and the conditioned medium induced cytopathic effect in the endothelial cells. In addition, N. fowleri can decrease the transendothelial electrical resistance by triggering the destabilization of the tight junction proteins claudin-5, occludin, and ZO-1 in a time-dependent manner. Furthermore, N. fowleri induced the expression of VCAM-1 and ICAM-1 and the production of IL-8, IL-1ß, TNF-α, and IL-6 as well as nitric oxide. We conclude that N. fowleri damaged the blood-brain barrier model by disrupting the intercellular junctions and induced the presence of inflammatory mediators by allowing the access of inflammatory cells to the olfactory bulbs.
Assuntos
Barreira Hematoencefálica/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/metabolismo , Células Endoteliais/metabolismo , Naegleria fowleri/metabolismo , Naegleria fowleri/patogenicidade , Proteínas de Junções Íntimas/metabolismo , Animais , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/patologia , Claudina-5/metabolismo , Cisteína Proteases/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Masculino , Meningoencefalite/parasitologia , Meningoencefalite/patologia , Camundongos , Mucosa/parasitologia , Mucosa/patologia , Ocludina/metabolismo , Ratos , Ratos Wistar , Trofozoítos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Conchas Nasais/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Naegleria fowleri is a protozoan that invades the central nervous system and causes primary amoebic meningoencephalitis. It has been reported that N. fowleri induces an important inflammatory response during the infection. In the present study, we evaluated the roles of Toll-like receptors in the recognition of N. fowleri trophozoites by human mucoepithelial cells, analyzing the expression and production of innate immune response mediators. After amoebic interactions with NCI-H292 cells, the expression and production levels of IL-8, TNF-α, IL-1ß, and human beta defensin-2 were evaluated by RT-PCR, ELISA, immunofluorescence, and dot blot assays, respectively. To determine whether the canonical signaling pathways were engaged, we used different inhibitors, namely, IMG-2005 for MyD88 and BAY 11-7085 for the nuclear factor NFkB. Our results showed that the expression and production of the pro-inflammatory cytokines and beta defensin-2 were induced by N. fowleri mainly through the canonical TLR4 pathway in a time-dependent manner.
Assuntos
Naegleria fowleri/imunologia , Naegleria fowleri/metabolismo , Receptores Toll-Like/metabolismo , Amebíase , Animais , Linhagem Celular , Citocinas/metabolismo , Defensinas/metabolismo , Células Epiteliais/imunologia , Humanos , Imunidade Inata , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Nitrilas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Trofozoítos/imunologia , Trofozoítos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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 is the aetiological agent of primary amoebic meningoencephalitis (PAM). This parasite invades its host by penetrating the olfactory mucosa. During the initial stages of infection, the host response is initiated by the secretion of mucus that traps the trophozoites. Despite this response, some trophozoites are able to reach, adhere to and penetrate the epithelium. In the present work, we evaluated the effect of mucins on amoebic adherence and cytotoxicity to Madin-Darby canine kidney (MDCK) cells and the MUC5AC-inducing cell line NCI-H292. We showed that mucins inhibited the adhesion of amoebae to both cell lines; however, this inhibition was overcome in a time-dependent manner. N. fowleri re-established the capacity to adhere faster than N. gruberi. Moreover, mucins reduced the cytotoxicity to target cells and the progression of the illness in mice. In addition, we demonstrated mucinolytic activity in both Naegleria strains and identified a 37 kDa protein with mucinolytic activity. The activity of this protein was inhibited by cysteine protease inhibitors. Based on these results, we suggest that mucus, including its major mucin component, may act as an effective protective barrier that prevents most cases of PAM; however, when the number of amoebae is sufficient to overwhelm the innate immune response, the parasites may evade the mucus by degrading mucins via a proteolytic mechanism.
Assuntos
Amebíase/imunologia , Mucinas/farmacologia , Naegleria fowleri/patogenicidade , Polissacarídeo-Liases/metabolismo , Amebíase/parasitologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Cães , Humanos , Rim/citologia , Rim/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucina-5AC/metabolismo , Mucinas/imunologia , Mucinas/toxicidade , Naegleria fowleri/crescimento & desenvolvimento , Naegleria fowleri/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
BACKGROUND: Primary amebic meningoencephalitis (PAM) is an emerging disease with a rapidly fatal outcome. Only eight reports of cured cases have appeared in the medical literature to date. METHODS: A 10-year-old boy developed PAM caused by Naegleria fowleri 1 week after swimming in an irrigation canal. He was admitted to our hospital after 9 h of severe headache and vomiting, fever, ataxic gait, mild confusion, and seizures were evident. Trophozoites were identified in the cerebrospinal fluid (CSF). Treatment with intravenous (i.v.) dexamethasone, amphotericin B, fluconaloze, and oral rifampicin was started. After several hours of conflicting clinical signs, recovery began, and on the third day he was conscious again. Hospital discharge occurred on day 23, after a normal brain CT scan. There was no sequel to the disease during the following 12 months. RESULTS: The amebas present in the CSF were identified and confirmed as N. fowleri after observation of wet mounts and of cultures seeded on 1.5% non-nutrient agar plates covered with Escherichia coli, vegetative and cystic forms, enflagellation experiments in distilled water at 98 degrees F, temperature tolerance testing and by indirect immunofluorescence using N. fowleri LEE antibody. The genotype was determined by PCR amplification and sequencing of the internal transcribed spacers (ITS) including the 5.8S rDNA. CONCLUSIONS: Early treatment of PAM by i.v. administration of amphotericin B and fluconazole, and oral administration of rifampicin can offer some hope of cure for this devastating disease.
Assuntos
Amebíase/tratamento farmacológico , Amebicidas/uso terapêutico , Anfotericina B/uso terapêutico , Fluconazol/uso terapêutico , Meningoencefalite , Naegleria fowleri , Rifampina/uso terapêutico , Amebíase/diagnóstico , Amebicidas/administração & dosagem , Anfotericina B/administração & dosagem , Animais , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Antifúngicos/administração & dosagem , Antifúngicos/uso terapêutico , Criança , Fluconazol/administração & dosagem , Humanos , Masculino , Meningoencefalite/diagnóstico , Meningoencefalite/tratamento farmacológico , Meningoencefalite/parasitologia , Naegleria fowleri/classificação , Naegleria fowleri/genética , Naegleria fowleri/metabolismo , Rifampina/administração & dosagemRESUMO
INTRODUCTION: Infections of the central nervous system by free-living amoeba are a rare phenomenon around the world. They present as a necrotising haemorrhagic acute meningoencephalitis, with a fatality rate close to 100% or as granulomatous amoebic encephalitis with a chronic progression. CASE REPORT: We describe a fatal case of primary amoebic meningoencephalitis produced by Naegleria fowleri in an 8-year-old child, with a history of immersion in a freshwater reservoir 52 hours before death. CONCLUSION: Laboratory studies carried out before death did not enable a diagnosis to be reached while the patient was alive. Diagnosis was made in the course of the post-mortem examination, where the presence of amoeba trophozoites was identified.