Abstract
Amphotericin B is an antibiotic used as the “gold standard” in the treatment of life-threatening fungal infections. Several molecular mechanisms have been proposed to explain exceptionally high effectiveness of amphotericin B in combating fungi. In the present work, we apply fluorescence lifetime imaging microscopy to track, step by step, modes of the toxic activity of amphotericin B towards a clinical strain of Candida albicans. The images recorded reveal that the antibiotic binds to cells in the form of the small aggregates characterized by a relatively short fluorescence lifetime (0.2 ns). Amphotericin B binds preferentially to the cell walls of mature cells but also to the plasma membranes of the daughter cells at the budding stage. The images recorded with the application of a scanning electron microscopy show that the antibiotic interferes with the formation of functional cell walls of such young cells. The results of imaging reveal the formation of the amphotericin B-rich extramembranous structures and also binding of the drug molecules into the cell membranes and penetration into the cells. These two modes of action of amphotericin B are observed in the time scale of minutes.
Highlights
Amphotericin B is an antibiotic used as the “gold standard” in the treatment of life-threatening fungal infections
Formation of extracellular bulk structures in the Candida cultures exposed to Amphotericin B (AmB), combined with pronounced morphological alterations of cells can clearly be visible by means of scanning electron microscopy (SEM, compare Figs 2 and 3)
We analysed the activity of molecules of antifungal antibiotic AmB towards Candida albicans with the application of the experimental approach that enables simultaneous monitoring of molecular organization and localization of the drug in the cells, based on specific spectroscopic signatures[11,13]
Summary
Amphotericin B is an antibiotic used as the “gold standard” in the treatment of life-threatening fungal infections. Formation of transmembrane pores able to act as ion channels interfering with the cell electrostasis is considered as one of the main mechanisms underlying the biological activity of AmB5–8 The fact that such intramembranous structures are more readily formed in the membranes containing ergosterol, a sterol of fungi, than in the case of cholesterol that is present in mammalian cells, is a central paradigm of AmB selectivity. A substantially different kind of the biological activity of AmB was demonstrated, relying in destabilization of biomembranes realized via extraction of sterols which were immobilized within extramembranous, two-component sterol-AmB sponge-like structures[9] Formation of such cholesterol-AmB structures, in the case of mammalian cells exposed to AmB, was demonstrated to have a potent protective effect against toxicity of the drug[10]. We apply a technique of fluorescence lifetime imaging microscopy (FLIM), which gives insight into the molecular organization and localization of AmB, to track and analyse different modes of the activity of the drug with respect to the popular, clinical strain of fungi: Candida albicans (referred to as Candida)
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