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JAC Advance Access originally published online on July 28, 2004
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Journal of Antimicrobial Chemotherapy 2004 54(3):603-608; doi:10.1093/jac/dkh385
JAC vol.54 no.3 © The British Society for Antimicrobial Chemotherapy 2004; all rights reserved

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

Antonella Lupetti1,2, Carlo P. J. M. Brouwer3, Heleen E. C. Dogterom-Ballering1, Sonia Senesi2, Mario Campa2, Jaap T. van Dissel1 and Peter H. Nibbering1,*

1 Department of Infectious Diseases, C5-P, Leiden University Medical Center (LUMC), P.O. Box 9600, 2300 RC Leiden; 3 AM-Pharma, Rumpsterweg, 6, 3981 AK Bunnik, The Netherlands; 2 Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università degli Studi di Pisa, Via S. Zeno, 35–39, 56127 Pisa, Italy

* Corresponding author. Tel: +31-71-5262620; Fax: +31-71-5266758; Email: p.h.nibbering{at}lumc.nl

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.

Keywords: lactoferrin peptide , mitochondrial Ca2+-uptake , mitochondrial membrane potential , reactive oxygen species production


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