RESUMO
An EcoRI fragment from the mitochondrial DNA of Acanthamoeba polyphaga was cloned and partly sequenced, and the conceptual translation product of the open reading frame (partial sequence) was found to have similarities with rp114, a ribosomal protein. Phylogenetic analysis based on the amino acid (aa) sequences of this conserved protein resolved four branches that consisted of: (1) eubacteria and the chloroplasts of algae and higher plants, (2) ciliate mitochondria, (3) Acanthamoeba, and (4) archaebacteria and the nuclei of eukaryotes. The groupings based on the rp114 aa sequences were consistent with the phylogenies derived by rRNA analysis of these organisms.
Assuntos
Acanthamoeba/genética , DNA Mitocondrial/genética , Filogenia , Proteínas Ribossômicas/genética , Acanthamoeba/química , Sequência de Aminoácidos , Animais , Archaea/química , Archaea/genética , Bactérias/química , Bactérias/genética , Sequência de Bases , Cloroplastos/química , Clonagem Molecular , Sequência Conservada , DNA Mitocondrial/análise , Células Eucarióticas/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Ribossômicas/química , Alinhamento de SequênciaRESUMO
PURPOSE: To identify host-tissue amoeba interactions that may be important in the pathogenesis of Acanthamoeba keratitis, the ability of the opportunistic pathogen Acanthamoeba polyphaga to bind various components of the extracellular matrix (collagen type IV, laminin, or fibronectin) was examined in vitro. METHODS: A polyphaga, isolated from a case of human amoebic keratitis, was used in the studies. In the experiments, 96-well plates were coated with 0-, 5-, 10-, 20-, or 50-micrograms/ml solutions of the basal lamina proteins laminin or collagen type IV, the extracellular matrix protein fibronectin, or casein (control). Amoeba were metabolically labeled with 35[S]-methionine, and 1x 10(4) labeled amoeba in phosphate buffered saline (PBS) were seeded per well and allowed to bind for 20 min. After washing with PBS, bound amoeba were solubilized with 1% sodium dodecyl sulphate (SDS) and scintillation counting was used to determine the number of bound amoeba. RESULTS: Counts from casein and protein-free controls were not significantly different from each other (P > 0.05). There was a significant increase in the binding of 35[S]-labeled A. polyphaga to collagen IV, laminin, and fibronectin over controls (P < 0.0001) and the binding was concentration-dependent. The rank order of binding was collagen > or = laminin >> fibronectin. Alpha-methyl-mannopyranoside, but not fucose, inhibited binding of labeled A. polyphaga to collagen IV, laminin, and fibronectin in a concentration-dependent manner. CONCLUSION: In summary, the binding assays show that Acanthamoeba bind preferentially to collagen, laminin, and fibronectin, in that order, and that the adherence process is inhibited by mannose.
Assuntos
Acanthamoeba/metabolismo , Colágeno/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Fucose/farmacologia , Metilmanosídeos/farmacologiaRESUMO
Benzimidazoles have been widely used since the 1960s as anthelmintic agents in veterinary and human medicine and as antifungal agents in agriculture. More recently, selected benzimidazole derivatives were shown to be active in vitro against two protozoan parasites, Trichomonas vaginalis and Giardia lamblia, and clinical studies with AIDS patients have suggested that microsporidia are susceptible as well. Here, we first present in vitro susceptibility data for T. vaginalis and G. lamblia using an expanded set of benzimidazole derivatives. Both parasites were highly susceptible to four derivatives, including mebendazole, flubendazole, and fenbendazole (50% inhibitory concentrations of 0.005 to 0.16 microgram/ml). These derivatives also had lethal activity that was time dependent: 90% of T. vaginalis cells failed to recover following a 20-h exposure to mebendazole at 0.17 microgram/ml. G. lamblia, but not T. vaginalis, was highly susceptible to five additional derivatives. Next, we examined in vitro activity of benzimidazoles against additional protozoan parasites: little or no activity was observed against Entamoeba histolytica, Leishmania major, and Acanthamoeba polyphaga. Since the microtubule protein beta-tubulin has been identified as the benzimidazole target in helminths and fungi, potential correlations between benzimidazole activity and beta-tubulin sequence were examined. This analysis included partial sequences (residues 108 to 259) from the organisms mentioned above, as well as the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi and the sporozoan Cryptosporidium parvum. beta-tubulin residues Glu-198 and, in particular, Phe-200 are strong predictors of benzimidazole susceptibility; both are present in Encephalitozoon spp. but absent in C. parvum.