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.

Introduction: Candidozyma auris (Cz. auris) has emerged globally, and diseases caused by it are associated with a mortality rate of 30–72%. This yeast is often multidrug-resistant and challenging to treat. A synthetic peptide, consisting of 11 amino acids of human lactoferrin (hLF1-11), offers a new therapy that is active against Candida albicans, non-albicans Candida yeasts, as well as Cz. auris. The current study examined the susceptibility of clinically relevant Candida species to hLF(1-11) in vitro and investigated the synergistic interaction of this peptide with fluconazole (FLU) and anidulafungin (ANI). Methods: Susceptibility of the yeasts to hLF(1-11) was tested with a microdilution method to determine minimum inhibitory concentrations (MICs). A total of 59 strains belonging to 16 species of Candida or Candidozyma were tested. The treatment cohort included 20 strains of Cz. auris originating from six different countries. Results: Mean MIC values of all susceptible strains ranged from 16.66 ± 6.46 μg/mL to 45.83 ± 10.21 μg/mL. There were no statistical differences in the susceptibility of hLF(1-11) for Cz. auris across geographic origins. In the combinatory tests, drugs acting together, the fractional inhibitory concentration indexes [FIC] < 1.0, showed a synergistic or additive effect on the efficacy of FLU and ANI when used in combination with hLF(1-11). [FIC] indexes 1–2 were interpreted as intermediate. MIC values in combinatory use were 1–2 titer steps lower than when used alone. Conclusions: hLF(1-11) inhibits the growth of yeasts that belong to the genus Candida, including Cz. auris. The combinatory use may be further investigated to treat infections caused by resistant yeasts.