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JAC Advance Access published online on July 28, 2004
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkh385
© 2004 by The British Society for Antimicrobial Chemotherapy
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Received January 21, 2004
Revised June 28, 2004
Accepted June 29, 2004

Original article

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 Lupetti 1, Carlo P. J. M. Brouwer 2, Heleen E. C. Dogterom-Ballering 3, Sonia Senesi 4, Mario Campa 4, Jaap T. van Dissel 3, Peter H. Nibbering 3*

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

* To whom correspondence should be addressed. E-mail: p.h.nibbering{at}lumc.nl.


  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.

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