RESUMO
Naegleria fowleri is a ubiquitous free-living amoeba that causes primary amoebic meningoencephalitis. As a part of the innate immune response at the mucosal level, the proteins lactoferrin (Lf) and lysozyme (Lz) are secreted and eliminate various microorganisms. We demonstrate that N. fowleri survives the individual and combined effects of bovine milk Lf (bLf) and chicken egg Lz (cLz). Moreover, amoebic proliferation was not altered, even at 24 h of co-incubation with each protein. Trophozoites' ultrastructure was evaluated using transmission electron microscopy, and these proteins did not significantly alter their organelles and cytoplasmic membranes. Protease analysis using gelatin-zymograms showed that secreted proteases of N. fowleri were differentially modulated by bLf and cLz at 3, 6, 12, and 24 h. The bLf and cLz combination resulted in the inhibition of N. fowleri-secreted proteases. Additionally, the use of protease inhibitors on bLf-zymograms demonstrated that secreted cysteine proteases participate in the degradation of bLf. Nevertheless, the co-incubation of trophozoites with bLf and/or cLz reduced the cytopathic effect on the MDCK cell line. Our study suggests that bLf and cLz, alone or together, inhibited secreted proteases and reduced the cytopathic effect produced by N. fowleri; however, they do not affect the viability and proliferation of the trophozoites.
RESUMO
Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity.
RESUMO
The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.
RESUMO
OBJECTIVES: Ameloblastoma is an odontogenic neoplasm of the mandible and maxilla with various histological types and subtypes. It has been reported that some ameloblastomas could arise from dentigerous cyst walls; thus, the development of ameloblastoma from dentigerous cysts may be due to differential protein expression. Our aim was to identify a membrane protein that is differentially expressed in ameloblastomas with respect to dentigerous cysts. METHODS: We analyzed the SDS-PAGE profiles of membrane proteins from ameloblastomas and dentigerous cysts. The protein in a band present in the ameloblastoma sample, but apparently absent in the dentigerous cyst sample was identified via mass spectrometry as the chaperonin Hsp60. We used western blotting and immunohistochemistry to analyze its overexpression and localization in ameloblastoma. RESULTS: We found a differential band of 95 kDa in the membrane proteins of ameloblastoma. In this band, the chaperonin Hsp60 was identified, and its overexpression was corroborated using western blotting and immunohistochemistry. Hsp60 was localized in the plasma membrane of all ameloblastoma samples studied; in addition, it was found in the cell nucleus of the plexiform subtype of conventional ameloblastoma. CONCLUSIONS: Our results suggest that Hsp60 may be involved in ameloblastoma development, and could therefore be a potential therapeutic target for ameloblastoma treatment.
Assuntos
Ameloblastoma , Chaperonina 60/genética , Cisto Dentígero , Proteínas Mitocondriais/genética , Tumores Odontogênicos , Ameloblastoma/genética , Chaperoninas , Humanos , Imuno-HistoquímicaRESUMO
The Naegleria are ubiquitous free-living amoebae and are characterized by the presence of three phases in their biological cycle: trophozoite, cyst and flagellate. Of this genus, only Naegleria fowleri has been reported as pathogenic to humans. The proteasome is a multi-catalytic complex and is considered to be the most important structure responsible for the degradation of intracellular proteins. This structure is related to the maintenance of cellular homeostasis and, in pathogenic microorganisms, to the modulation of their virulence. Until now, the proteasome and its function have not been described for the Naegleria genus. In the current study, using bioinformatic analysis, protein sequences homologous to those reported for the subunits of the 20S proteasome in other organisms were found, and virtual modelling was used to determine their three-dimensional structure. The presence of structural and catalytic subunits of the 20S proteasome was detected by Western and dot blot assays. Its localization was observed by immunofluorescence microscopy to be mainly in the cytoplasm, and a leading role of the chymotrypsin-like catalytic activity was determined using fluorogenic peptidase assays and specific proteasome inhibitors. Finally, the role of the 20S proteasome in the proliferation and differentiation of Naegleria genus trophozoites was demonstrated.
Assuntos
Naegleria fowleri/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Proliferação de CélulasRESUMO
INTRODUCTION AND OBJECTIVES: Curcumin, a polyphenol, is a natural compound that has been widely studied as a hepatoprotector; however, only a few studies have examined its ability to reduce fibrosis in previously established cirrhosis. The objective of this study was to investigate whether curcumin could reduce carbon tetrachloride (CCl4)-induced fibrosis and if so, to determine the action mechanisms involved in the reduction process. MATERIALS AND METHODS: CCl4 was administered to male Wistar rats (400â¯mg/kg, three times a week, i. p.) for 12 weeks; curcumin (100â¯mg/kg body weight twice per day, p. o.) was administered from week 9-12 of CCl4 treatment. Biochemical markers of hepatic injury and oxidative stress were evaluated. Hematoxylin and eosin, Masson's trichrome stains, transmission electron microscopy; immunohistochemistry, and zymography assays were carried out. Moreover, Smad3 and α-SMA mRNA and protein levels were studied. Western blotting by TGF-ß, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, pSmad3, and pJNK proteins was developed. RESULTS AND CONCLUSIONS: Curcumin reduced liver damage, oxidative stress, fibrosis, and restored normal activity of MMP-9 and MMP-2. Besides, curcumin restored NF-κB, IL-1, IL-10, TGF-ß, CTGF, Col-I, MMP-13, and Smad7 protein levels. On the other hand, curcumin decreased JNK and Smad3 phosphorylation. Furthermore, curcumin treatment decreased α-SMA and Smad3 protein and mRNA levels. Curcumin normalized GSH, and NF-κB, JNK-Smad3, and TGF-ß-Smad3 pathways, leading to a decrement in activated hepatic stellate cells, thereby producing its antifibrotic effects.
Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Curcumina/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Proteína Smad3/metabolismo , Proteína Smad7/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citocinas/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/ultraestrutura , Fígado/metabolismo , Fígado/ultraestrutura , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Ratos Wistar , Transdução de SinaisRESUMO
Currently, there is growing interest in the identification and purification of microbial lectins due to their involvement in the pathogenicity mechanisms of pathogens, such as Entamoeba histolytica and free-living amoebae. The Gal/GalNAc lectin from E. histolytica participates in adhesion, cytotoxicity and regulation of immune responses. Furthermore, mannose- and galactose-binding protein have been described in Acanthamoeba castellanii and Balamuthia mandrillaris, respectively and they also contribute to host damage. Finally, in Naegleria fowleri, molecules containing mannose and fucose are implicated in adhesion and cytotoxicity. Considering their relevance in the pathogenesis of the diseases caused by these protozoa, lectins appear to be promising targets in the diagnosis, vaccination and treatment of these infections.
Assuntos
Amoeba/efeitos dos fármacos , Entamoeba histolytica/efeitos dos fármacos , Lectinas/farmacologia , Fatores de Virulência , Amebíase/diagnóstico , Animais , Balamuthia mandrillaris , Entamebíase/diagnóstico , Entamebíase/tratamento farmacológico , Entamebíase/parasitologia , Glicoconjugados , Glicoproteínas , Interações Hospedeiro-Parasita , Humanos , Naegleria fowleri , VacinaçãoRESUMO
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
Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5â¯T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.