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AmphotericinB mediates killing in C.neoformans through induction of oxidative burst rather than through pore formation at the membrane

Abstract number: O295

Zaragoza O., Scorzoni L., Sangalli Leite F., Gianinni M.J., Rodríguez-Tudela J.L., Cuenca-Estrella M.

Objectives: Amphotericin B (AmB) is an antifungal drug widely used for the treatment of fungal infections, such as cryptococcal meningitis, which is caused by the encapsulated fungal pathogen Cryptococcus neoformans. AmB binds to ergosterol and forms pores at the membrane, resulting in cell death. However, other reports indicate that AmB also acts as an oxidant. In this work we have studied the effect on AmB on C. neoformans.

Methods: We have compared three viability methods: XTT assay, prodium iodide staining, and colony forming units enumeration. XTT is reduced in the mitochondria by living cells, producing a compound that is quantified spetrophotometrically. Propidium iodide is a DNA-binding fluorescent compound that only penetrates in the cells once they lose the membrane integrity. Finally, CFUs enumeration estimates the ability of the cells to replicate and form viable colonies.

Results: While AmB inhibited the formation of colonies at concentrations above 0.5–1 mg/L, the cells did not become permeable to propidium iodide in the same conditions, suggestin that AmB has other effects on C. neoformans different than pore formation. When viability was measured using the XTT assay, a correlation with the CFUs results was observed, confirming that AmB exerts its killing effect intracellularly. However, we also observed a "paradoxical effect" in which cells treated with high AmB concentrations (4–16 mg/L) produced higher levels of reduced XTT than cells treated with intermediate AmB concentrations (0.25–1 mg/L). Since this "paradoxical effect did not correlate with CFUs appearance, we argued that it might reflect an induction of the electron transfer in the mitochondria, as a consequence of an increased oxidative burst induced by AmB. So we measured the amount of reactive oxygen species (ROS) in the cells using dihydrofluorescein, a compound that when is attacked by free radicals releases fluorescence. Our results demonstrated that AmB induced a strong release of ROS in the cells, which correlates with the metabolic inactivation measured by the XTT assay.

Conclusions: Although AmB can bind to ergosterol and produce pores at the membrane, our results indicate that this antifungal drug induces killing in C. neoformans mainly through an induction of a strong oxidative burst. These findings confirm that Am B has multiple effects on the cells, which suggests an explanation for the low antifungal resistance to this compound observed among clinical isolates.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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