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, 3539, 56127 Pisa, Italy
Received 21 January 2004; returned 22 March 2004; revised 28 June 2004; accepted 29 June 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: The effect of compounds interfering with Ca2+ homeostasis on the hLF(111)-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(111)-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(111)-induced candidacidal activity. Moreover, hLF(111) 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(111)-induced candidacidal activity.
Keywords: lactoferrin peptide , mitochondrial Ca2+-uptake , mitochondrial membrane potential , reactive oxygen species production
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study was undertaken to gain more insight into the role of Ca2+ in mitochondrial activation (changes in membrane potential, reactive oxygen species production) preceding mitochondrial dysfunctioning and the subsequent death of C. albicans upon exposure to hLF(111).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The synthetic peptide corresponding to residues 111 (GRRRRSVQWCA; 1374 Da) of human lactoferrin, further referred to as hLF(111), was prepared and purified as described previously.19 The purity of the peptide exceeded 88%, as determined by reversed-phase high performance liquid chromatography. Stocks of peptide at a concentration of 1 mg/mL of 0.01% acetic acid (HAc; pH 3.7) were stored at 20°C and dried in a Speed-Vac immediately before use (Savant Instruments Inc., Farmingdale, NY, USA).
Chemicals
CaCl2.2H2O was purchased from Merck (Darmstadt, Germany), Ruthenium Red and ethylene glycol-bis(ß-aminoethyl-ether)-N,N,N1,N1-tetraacetic acid (EGTA) from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Standard oxalate solution and ionomycin were obtained from Sigma Chemical Co. (St Louis, MO, USA). The oxalate standard solution and stocks of EGTA (1 M) and ionomycin (100 mM), prepared in water were stored at 4°C until use. A stock of Ruthenium Red (10 mM) was prepared in 50% DMSO and stored at 20 °C. Calcium chloride (50 mM) was freshly prepared in 10 mM sodium phosphate buffer, pH 7.4.
Source of C. albicans strain
Fluconazole-resistant C. albicans strain Y01-19 was purchased from Pfizer Inc. (Groton, CT, USA). Fluconazole resistance (MIC > 256 mg/L) was evaluated with the Etest (Oxoid Unipath Ltd., Basingstoke, UK). Yeasts were cultured overnight in Sabouraud broth (Oxoid) at 37°C and subcultured for 2.5 h on a rotary wheel at 37°C.
Treatment of C. albicans with inhibitors
Calcium chloride (50 mM) or EGTA (10 mM) were added simultaneously with the peptide to C. albicans. In addition, C. albicans cells were pre-incubated with ionomycin (10 µM up to 50 mM), Ruthenium Red (30 µM) or oxalate (340 µM), for 15 min at 37°C before addition of the peptide. These optimal concentrations were established in preliminary experiments to avoid toxic effects of these compounds on C. albicans.
Assay for candidacidal activity of hLF(111)
An in vitro assay was used to assess the candidacidal activity of hLF(111).6 Briefly, yeast cells were harvested in mid-log phase by centrifugation (1500 g, 10 min), washed twice in sodium phosphate buffer, and diluted to a concentration of 1 x 106 cfu/mL of buffer. Next, equal volumes of this suspension and various concentrations of hLF(111) were mixed in small plastic snap-cap vials. After incubation for 2 h at 37°C, the vials were transferred onto ice and the number of viable blastoconidia was determined by plating serial dilutions of each sample on Sabouraud agar. Results, expressed as cfu C. albicans per mL, are means plus standard deviation (S.D.).
Measurement of 45Ca2+ accumulation
Accumulation of Ca2+ in C. albicans was measured as previously described.17,20 In brief, yeast cells grown in yeast peptone dextrose (YPD) medium (pH 5.5) at 30°C were harvested in mid-log phase by centrifugation (1500 g, 10 min) and 5 x 105 cfu were resuspended in 1.5 mL of fresh YPD medium supplemented with 20 µCi of 45CaCl2 (Amersham Biosciences Europe GmbH, Freiburg, Germany) per mL. Next, these C. albicans cells were exposed at 30°C to 17 µM hLF(111) for various intervals. The reaction was stopped by transferring the cells onto melting ice and C. albicans were harvested onto 2.4 cm GF/F filters (Whatman International Ltd, Kent, UK), washed with ice-cold buffer [10 mM CaCl2, 5 mM HEPESNaOH (Sigma); pH 6.5], dried at 90°C for 1 h, placed in scintillation vials, and processed for liquid scintillation counting in a Packard 1600 TR liquid scintillation counter using OptiFluor (Packard) scintillation cocktail after addition of 10 mL of Ultima Flo-M (Packard BioScience B.V., Groningen, The Netherlands). Results of the total cell-associated radioactivity are expressed as disintegrations per min (dpm).
Assay for mitochondrial membrane potential
The positively charged fluorescent probe rhodamine 123 (Molecular Probes, Eugene, OR, USA) was used to measure the mitochondrial membrane potential in C. albicans.6 Briefly, C. albicans in mid log-phase was resuspended in 1 mM potassium phosphate buffer, pH 7.0 and pre-incubated for 10 min at 37°C with 10 µM rhodamine 123 in the same buffer. After two washes with buffer, C. albicans cells were treated for 10 min at 37°C with 17 µM hLF(111) and then analysed on a fluorescence-activated cell sorter (FACS) FACSCalibur (Becton & Dickinson, San José, CA, USA) equipped with an argon laser at 488 nm. Rhodamine 123 median fluorescence intensities (MFI) were measured in the second channel. Data acquisition and analysis were controlled with the CellQuest Pro software and hardware interface. Results, expressed as the percentage of the MFI for hLF(111)-exposed C. albicans, are means ± S.D.
Measurement of reactive oxygen species production
2',7'-Dichlorofluorescein diacetate (Eastman Kodak Company, Rochester, NY, USA) was used to measure reactive oxygen species production by C. albicans as previously described.12 In brief, C. albicans cells were harvested in mid-log phase, washed twice as described above, and then diluted to a concentration of 2 x 106 cfu/mL of sodium phosphate buffer. Next, C. albicans cells were pre-incubated for 15 min at 37°C with 100 mM of 2',7'-dichlorofluorescein diacetate then treated for 15 min at 37°C with various concentrations of hLF(111). Immediately before use, 2',7'-dichlorofluorescein diacetate (100 mM) was dissolved in DMSO and further diluted in buffer. The fluorescence of DCF was measured on the FACSCalibur. Results are expressed as MFI + S.D.
Statistical analysis
Differences between the values for hLF(111)-treated and untreated C. albicans were analysed by the MannWhitney U-test. The level of significance was set at P < 0.05. Correlation between the accumulation of Ca2+ in C. albicans and the exposure to 17 µM hLF(111) in time was calculated using the Pearson correlation test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate whether Ca2+ is essential for the candidacidal activity of hLF(111), the effect of 10 mM EGTA on the candidacidal activity of this peptide was determined in sodium phosphate buffer, pH 7.4. The results revealed that this calcium chelator completely blocked (P < 0.05) the candidacidal activity of the peptide (Figure 1). In addition, the combination of the ionophore ionomycin (10 µM) and CaCl2 (50 mM) increased (P < 0.05) the candidacidal activity induced by the highest concentration of hLF(111) tested. The number of C. albicans cells surviving upon treatment with 67 µM hLF(111) amounted to 5.1 ± 2.6 x 103 and after exposure to the combination to 1.4 ± 0.8 x 103 cfu/mL (n=4), suggesting that a rise in [Ca2+]cyt plays a role in the hLF(111)-induced candidacidal activity.
|
45Ca2+ accumulation was measured in C. albicans upon exposure to hLF(111). The results revealed a linear increase (r=0.791; P < 0.001) in Ca2+ accumulation in C. albicans upon exposure to 17 µM hLF(111) reaching a three-fold increase at 2 h (Figure 2), whereas no increase was found during this period of analysis in C. albicans not exposed to the peptide.
|
Since effects on mitochondria are essential for the candidacidal activity of hLF(111), the action of Ruthenium Red, which inhibits the Ca2+-uptake by mitochondria21 and the voltage-sensitive Ca2+ release from internal stores,22 on the killing of C. albicans was evaluated. The results revealed that this inhibitor blocked (P < 0.05) the candidacidal activity of hLF(111) (Figure 3).
|
To investigate whether Ca2+ released by intracellular stores is involved in the candidacidal activity of hLF(111), the effects of oxalate, which precipitates Ca2+ in intracellular organelles,23 and high extracellular Ca2+ (in the mM range), which inhibits Ca2+ release from intracellular stores in various cell types,24 were evaluated. The results revealed that oxalate partially inhibited (P < 0.05), whereas high Ca2+ completely blocked the hLF(111)-induced killing of C. albicans (Figure 4), indicating that Ca2+ release from intracellular stores is crucial in the candidacidal activity of hLF(111).
|
The role of mitochondrial Ca2+-uptake and Ca2+ release from intracellular stores in the hLF(111)-induced changes in the mitochondrial membrane potential was further investigated using rhodamine 123 and FACS analysis.6 The results revealed that (i) the hLF(111) peptide induced a significant (P < 0.05) increase in rhodamine 123 fluorescence in C. albicans, compared with unexposed cells, and that (ii) Ruthenium Red, oxalate, high extracellular CaCl2, and EGTA completely blocked (P < 0.05) the hLF(111)-induced change in the mitochondrial rhodamine 123 staining (Table 1), suggesting that mitochondrial Ca2+-uptake and Ca2+ release from intracellular stores are essential for the hLF(111)-induced changes in the mitochondrial membrane potential.
|
Next, the effect of previously mentioned pharmacological agents on the hLF(111)-induced reactive oxygen species production was studied. The results revealed that Ruthenium Red, oxalate, high extracellular CaCl2 and EGTA completely blocked reactive oxygen species production (P < 0.05) by C. albicans upon exposure to hLF(111) (Figure 5).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although assessment of changes in [Ca2+]cyt in C. albicans with classical fluorescent probes, e.g. Fura-2/AM,25 failed (most likely due to the very low [Ca2+]cyt in yeasts), an indirect estimation of changes in Ca2+ content was obtained by measuring the accumulation of 45Ca2+ in C. albicans from extracellular medium, which is essential for the replenishment of Ca2+-depleted organelles in C. albicans.26,27 Our results showed that the hLF(111) peptide stimulates a linear increase in cell-associated Ca2+ in time, reaching a three-fold increase in the cellular Ca2+ content at 2 h, whereas no increase in Ca2+ accumulation was found in cells not exposed to the peptide or exposed to a control peptide (data not shown).
Together, the present data indicate that the hLF(111) peptide causes intracellular stores to release Ca2+ into the cytosol as well as an influx of Ca2+ from the extracellular medium; this may result in an influx of Ca2+ into mitochondria, a change in the mitochondrial membrane potential and the formation of the reactive oxygen species. Further studies are needed to prove that the hLF(111) peptide-induced Ca2+ influx into mitochondria may lead to the destruction of the energized mitochondria.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Helmerhorst, E. J., Breeuwer, P., van't Hof, W. et al. (1999). The cellular target of histatin 5 on Candida albicans is the energized mitochondrion. Journal of Biological Chemistry 274, 728691.
3
.
Edgerton, M., Koshlukova, S. E., Lo, T. E. et al. (1998). Candidacidal activity of salivary histatins. Identification of a histatin 5-binding protein on Candida albicans. Journal of Biological Chemistry 273, 2043847.
4
.
Gyurko, C., Lendenmann, U., Troxler, R. F. et al. (2000). Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5. Antimicrobial Agents and Chemotherapy 44, 34854.
5
.
Koshlukova, S. E., Araujo, M. W. B., Baev, D. et al. (2000). Released ATP is an extracellular cytotoxic mediator in salivary histatin 5-induced killing of Candida albicans. Infection and Immunity 68, 684856.
6
.
Lupetti, A., Paulusma-Annema, A., Welling, M. M. et al. (2000). Candidacidal activities of human lactoferrin peptides derived from the N terminus. Antimicrobial Agents and Chemotherapy 44, 325763.
7
.
Lupetti, A., Paulusma-Annema, A., Welling, M. M. et al. (2003). Synergistic activity of the N-terminal peptide of human lactoferrin and fluconazole against Candida species. Antimicrobial Agents and Chemotherapy 47, 2627.
8 . Brock, J. (1995). Lactoferrin: a multifunctional immunoregulatory protein? Immunology Today 16, 4179.[CrossRef][ISI][Medline]
9 . Bullen, J. J. (1981). The significance of iron in infection. Review of Infectious Diseases 3, 112738.[ISI][Medline]
10 . Bellamy, W., Takase, M., Yamauchi, K. et al. (1992). Identification of the bactericidal domain of lactoferrin. Biochimica et Biophysica Acta 1121, 1306.[ISI][Medline]
11
.
Helmerhorst, E. J., Troxler, R. F. & Oppenheim, F. G. (2001). The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species. Proceedings of the National Academy of Sciences, USA 98, 1463742.
12
.
Lupetti, A., Paulusma-Annema, A., Senesi, S. et al. (2002). Internal thiols and reactive oxygen species in candidacidal activity exerted by an N-terminal peptide of human lactoferrin. Antimicrobial Agents and Chemotherapy 46, 16349.
13 . Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature 415, 38995.[CrossRef][ISI][Medline]
14
.
Denis, V. & Cyert, M. S. (2002). Internal Ca2+ release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue. Journal of Cell Biology 156, 2934.
15 . Halachmi, D. & Eilam, Y. (1989). Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1. FEBS Letters 256, 5561.[CrossRef][ISI][Medline]
16
.
Strayle, J., Pozzan, T. & Rudolph, H. K. (1999). Steady-state free Ca2+ in the yeast endoplasmic reticulum reaches only 10 µM and is mainly controlled by the secretory pathway pump Pmr1. EMBO Journal 18, 473343.
17 . Cunningham, K. W. & Fink, G. R. (1996). Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Molecular and Cellular Biology 16, 222637.[Abstract]
18 . Miseta, A., Kellermayer, R., Aiello, D. P. et al. (1999). The vacuolar Ca2+/H+ exchanger Vcx1p/Hum1p tightly controls cytosolic Ca2+ levels in Saccharomyces cerevisiae. FEBS Letters 451, 1326.[CrossRef][ISI][Medline]
19 . de Koster, H. S., Amons, R. & Benckhuijsen, W. E. (1995). The use of dedicated peptide libraries permits the discovery of high affinity binding peptides. Journal of Immunological Methods 187, 17988.[CrossRef][ISI][Medline]
20
.
Bonilla, M., Nastase, K. K. & Cunningham, K. W. (2002). Essential role of calcineurin in response to endoplasmic reticulum stress. EMBO Journal 21, 234353.
21
.
Jambrina, E., Alonso, R., Alcalde, M. et al. (2003). Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death. Journal of Biological Chemistry 278, 1413445.
22
.
Calvert, C. M. & Sanders, D. (1995). Inositol trisphosphate-dependent and -independent Ca2+ mobilization pathways at the vacuolar membrane of Candida albicans. Journal of Biological Chemistry 270, 727280.
23 . Raeymaekers, L., Wuytack, F., Eggermont, J. et al. (1983). Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum. Biochemical Journal 210, 31522.[ISI][Medline]
24 . Bezprozvanny, I., Watras, J. & Ehrlich, B. E. (1991). Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 7514.[CrossRef][ISI][Medline]
25 . Alfonso, A., De la Rosa, L. A., Vieytes, M. R. et al. (2003). Dimethylsphingosine increases cytosolic calcium and intracellular pH in human T lymphocytes. Biochemical Pharmacology 65, 46578.[CrossRef][ISI][Medline]
26
.
Locke, E. G., Bonilla, M., Liang, L. et al. (2000). A homolog of voltage-gated Ca2+ channels stimulated by depletion of secretory Ca2+ in yeast. Molecular and Cellular Biology 20, 668694.
27 . Putney, J. W., Broad, L. M., Braun, F. J. et al. (2001). Mechanisms of capacitative calcium entry. Journal of Cell Science 114, 22239.[ISI][Medline]
|