RESUMO
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.
Assuntos
Acanthamoeba/genética , Acanthamoeba/patogenicidade , Genótipo , Acanthamoeba/enzimologia , Animais , Cães , Células Epiteliais/parasitologia , Células Epiteliais/patologia , Células Madin Darby de Rim Canino , Serina Proteases/metabolismo , Proteínas de Junções Íntimas/metabolismoRESUMO
Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.
Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/metabolismo , Serina Proteases/metabolismo , Acanthamoeba/genética , Ceratite por Acanthamoeba/parasitologia , Animais , Cultura Axênica , Caseínas/análise , Linhagem Celular , Cães , Genótipo , Humanos , Células Madin Darby de Rim Canino , Trofozoítos/metabolismoRESUMO
Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.
Assuntos
Ceratite por Acanthamoeba/terapia , Acanthamoeba/efeitos dos fármacos , Clorexidina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Proteínas de Protozoários/antagonistas & inibidores , Trofozoítos/efeitos dos fármacos , Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/patologia , Animais , Córnea/efeitos dos fármacos , Córnea/parasitologia , Córnea/patologia , Modelos Animais de Doenças , Esquema de Medicação , Composição de Medicamentos/métodos , Quimioterapia Combinada , Análise Fatorial , Ácidos Graxos Monoinsaturados/química , Regulação da Expressão Gênica , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Humanos , Lipossomos/metabolismo , Fosfatidiletanolaminas/química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Polietilenoglicóis/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Trofozoítos/enzimologia , Trofozoítos/patogenicidadeRESUMO
Current treatments for Acanthamoeba keratitis are unspecific. Because of the presence of the resilient cyst form of the parasite, the infection is persistent. Silencing the key protein of cyst formation, glycogen phosphorylase, has shown potential for reducing encystment processes of the Acanthamoeba trophozoite. However, a suitable carrier to protect and deliver siRNA sequences is still needed. DOTAP: DOPE:DSPE-PEG liposomes were prepared by three different techniques and used to associate a therapeutic siRNA sequence. Liposomes prepared by film hydration followed by membrane extrusion were considered the most adequate ones with average size of 250 nm and zeta potential of +45 mV, being able to associate siRNA for at least 24 hr in culture medium. siRNA-liposomes could inhibit up to 66% of the encystment process. Cell viability studies demonstrated MTT reduction capacity higher than 80% after 3 hr incubation with this formulation. After 24 hr of incubation, LDH activity ranged for both the formulations from around 4% to 40%. In vivo tolerance studies in mice showed no macroscopic alteration in the eye structures up to 24 hr after eight administrations during 1 day. Histological studies showed regular tissue architecture without any morphological alteration. Overall, these results suggest that the formulations developed are a promising new strategy for the treatment of ocular keratitis caused by Acanthamoeba spp.
Assuntos
Acanthamoeba/efeitos dos fármacos , Córnea/efeitos dos fármacos , Lipossomos/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Acanthamoeba/enzimologia , Acanthamoeba/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Córnea/metabolismo , Córnea/parasitologia , Córnea/patologia , Olho/efeitos dos fármacos , Olho/metabolismo , Olho/parasitologia , Olho/patologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Humanos , Lipossomos/toxicidade , Masculino , Camundongos , Tamanho da Partícula , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA Interferente Pequeno/químicaRESUMO
Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.
Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/virologia , Interações Hospedeiro-Parasita , Mimiviridae/crescimento & desenvolvimento , Serina Proteases/biossíntese , Esporos de Protozoários/crescimento & desenvolvimento , Acanthamoeba/crescimento & desenvolvimentoRESUMO
The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue destruction. An interestingly finding was that no lysis of corneal tissue was observed as it was previously suggested. These results, together with previous studies, allow us to conclude that the invasion and disruption of corneal tissue is performed by the penetration of the amoebae through cell junctions, either by the action of proteases promoting cellular separation but not by their destruction and/or a mechanical effect exerted by amoebae. Therefore, contact-dependent mechanisms in Acanthamoeba pathogenesis are more relevant than it has been previously considered. This is supported because the phagocytosis of recently detached cells as well as those attached to the corneal epithelium leads to the modification of the cellular architecture facilitating the migration and destruction of deeper layers of the corneal epithelium.
Assuntos
Acanthamoeba , Amebíase , Epitélio Corneano , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Amebíase/enzimologia , Amebíase/patologia , Animais , Cricetinae , Cães , Epitélio Corneano/metabolismo , Epitélio Corneano/parasitologia , Epitélio Corneano/ultraestrutura , Junções Intercelulares/metabolismo , Junções Intercelulares/parasitologia , Junções Intercelulares/ultraestrutura , Células Madin Darby de Rim Canino , Masculino , MesocricetusRESUMO
Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.
Assuntos
Ceratite por Acanthamoeba/patologia , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/enzimologia , Proteínas de Protozoários/metabolismo , Serina Proteases/metabolismo , Acanthamoeba/isolamento & purificação , Adulto , Eletroforese , Humanos , Pessoa de Meia-Idade , Peso Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Índice de Gravidade de Doença , Estatística como Assunto , 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
Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.