Release of calcium from intracellular stores and subsequent uptake by mitochondria are essential for the candidacidal activity of an N-terminal peptide of human lactoferrin

Abstract

Objectives: Earlier studies showed that mitochondrial damage is a hallmark of the candidacidal activity of an N-terminal peptide of human lactoferrin, further referred to as hLF(1-11). Since uptake of Ca2+ by mitochondria may be essential for their activation, the aim of this study was to define the role of Ca2+ in killing of Candida albicans by the hLF(1-11) peptide. Methods: The effect of compounds interfering with Ca2+ homeostasis on the hLF(1-11)-induced candidacidal activity, changes in mitochondrial membrane potential, and reactive oxygen species production were evaluated using a killing assay, rhodamine 123 staining, and 2′,7′-dichlorofluorescein diacetate, respectively. The increase in cellular Ca2+ content was measured using 45Ca2+. Results: Our results revealed that Ruthenium Red, which inhibits the mitochondrial Ca2+-uniporter and the voltage-sensitive Ca2+ release from internal stores, blocked (P < 0.05) the hLF(1-11 -induced candidacidal activity as well as changes in the membrane potential of mitochondria, and reactive oxygen species production. Oxalate, which precipitates Ca2+ in intracellular organelles, decreased (P < 0.05) the peptide-induced changes in the membrane potential of mitochondria, reactive oxygen species production, and candidacidal activity. Furthermore, the Ca2+ ionophore ionomycin combined with high CaCl2 concentrations enhanced the hLF(1-11)-induced candidacidal activity. Moreover, hLF(1-11) caused an influx of Ca2+ from the extracellular medium into C. albicans reaching a three-fold increase at 2 h, whereas no increase was found in unexposed cells. In agreement, the Ca2+-chelator EGTA blocked the peptide-induced candidacidal activity. Conclusions: Ca2+ release from intracellular stores, probably through subsequent mitochondrial Ca2+ uptake, is essential for the hLF(1-11)-induced candidacidal activity. © The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.