RESUMO
Psd1 is a plant defensin that has antifungal activity against several pathogenic and nonpathogenic fungi. Previous analysis of Psd1 chemical shift perturbations by nuclear magnetic resonance (NMR) spectroscopy demonstrated that this defensin interacts with phospholipids and the sphingolipid glucosylceramide isolated from Fusarium solani (GlcCer(Fusarium solani)). In this study, these interactions were evaluated by real-time surface plasmon resonance (SPR) analysis. The data obtained demonstrated that Psd1 could bind more strongly to small unilamellar vesicles (SUV)-containing GlcCer(Fusarium solani) than to SUV that was composed of phosphatidylcholine (PC) alone or was enriched with GlcCer that had been isolated from soybeans. An increase in the SPR response after cholesterol or ergosterol incorporation in PC-SUV was detected; however, SUV composed of PC:Erg (7:3; molar:molar) became unstable in the presence of Psd1, suggesting membrane destabilization. We also observed a lack of Psd1 internalization in Candida albicans strains that were deficient in the glucosyl ceramide synthase gene. Together, these data indicate that GlcCer is essential for Psd1 anchoring in the fungal plasma membrane as well as internalization.
Assuntos
Candida albicans/fisiologia , Defensinas/metabolismo , Glucosilceramidas/metabolismo , Lipossomos/metabolismo , Proteínas de Plantas/metabolismo , Ressonância de Plasmônio de Superfície , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Defensinas/farmacologia , Endocitose/efeitos dos fármacos , Cinética , Testes de Sensibilidade Microbiana , Microscopia Confocal , Fosfatidilcolinas/metabolismo , Proteínas de Plantas/farmacologiaRESUMO
The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100oC for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction