title: Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst.
abstract: We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.
authors.affiliation: Mycology Reference Laboratory, National Centre for Microbiology, Instituto de Salud Carlos III, Crta Majadahonda-Pozuelo Km 2, Majadahonda 28220, Madrid, Spain.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Sangalli-Leite
authors.forename: Liliana
authors.initials: L
authors.lastname: Scorzoni
authors.forename: Ana Cecilia
authors.initials: AC
authors.lastname: Mesa-Arango
authors.forename: Celia
authors.initials: C
authors.lastname: Casas
authors.forename: Enrique
authors.initials: E
authors.lastname: Herrero
authors.forename: Maria José Soares Mendes
authors.initials: MJ
authors.lastname: Gianinni
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Rodríguez-Tudela
authors.forename: Manuel
authors.initials: M
authors.lastname: Cuenca-Estrella
authors.forename: Oscar
authors.initials: O
authors.lastname: Zaragoza
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology*; D000935:Antifungal Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D015169:Colony Count, Microbial; D003455:Cryptococcus neoformans / Q000187:drug effects* / Q000254:growth & development / Q000502:physiology; D008826:Microbial Sensitivity Tests; D010084:Oxidation-Reduction; D011419:Propidium / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects*; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; D011419:Propidium; D000666:Amphotericin B