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.
OBJECTIVE
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 Ca(2+) by mitochondria may be essential for their activation, the aim of this study was to define the role of Ca(2+) in killing of Candida albicans by the hLF(1-11) peptide.


METHODS
The effect of compounds interfering with Ca(2+) 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 Ca(2+) content was measured using (45)Ca(2+).


RESULTS
Our results revealed that Ruthenium Red, which inhibits the mitochondrial Ca(2+)-uniporter and the voltage-sensitive Ca(2+) 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 Ca(2+) 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 Ca(2+) ionophore ionomycin combined with high CaCl(2) concentrations enhanced the hLF(1-11)-induced candidacidal activity. Moreover, hLF(1-11) caused an influx of Ca(2+) 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 Ca(2+)-chelator EGTA blocked the peptide-induced candidacidal activity.


CONCLUSIONS
Ca(2+) release from intracellular stores, probably through subsequent mitochondrial Ca(2+) uptake, is essential for the hLF(1-11)-induced candidacidal activity.
Lupetti
Antonella
A
Department of Infectious Diseases, C5-P, Leiden University Medical Center (LUMC), P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Brouwer
Carlo P J M
CP
Dogterom-Ballering
Heleen E C
HE
Senesi
Sonia
S
Campa
Mario
M
Van Dissel
Jaap T
JT
Nibbering
Peter H
PH
D000935:Antifungal Agents; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002132:Calcium Radioisotopes; D002176:Candida albicans / Q000187:drug effects*; D002614:Chelating Agents / Q000494:pharmacology; D025141:Drug Resistance, Fungal; D004533:Egtazic Acid / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007781:Lactoferrin; D008564:Membrane Potentials / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D008928:Mitochondria / Q000378:metabolism*; D010070:Oxalates / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology
D000935:Antifungal Agents; D002132:Calcium Radioisotopes; D002614:Chelating Agents; D010070:Oxalates; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; C466658:lactoferrin (1-11), human; D012430:Ruthenium Red; D004533:Egtazic Acid; D015759:Ionomycin; D007781:Lactoferrin; D002122:Calcium Chloride; D002118:Calcium