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RESEARCH REPORT |
1 Department of Oral Biology and
2 Restorative Dentistry, School of Dental Medicine, State University of New York at Buffalo, 310 Foster Hall, SUNY at Buffalo Main Street Campus, 3435 Main Street, Buffalo, NY 14214, USA;
* corresponding author, edgerto{at}buffalo.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Candida albicans • histatin • saliva • calcium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Although Hsts possess significant in vitro antifungal activity, the extent of candidacidal activity in saliva in vivo is less clear. Whole or glandular salivas do not exhibit the level of candidacidal activity that should be provided according to the calculated concentrations of histatins. Thus, the concept of salivary "masking" has been proposed to account for the discrepancy between in vitro and in vivo activity of histatins (Flora et al., 2002). Nearly all in vitro studies of the candidacidal action of Hsts have been performed in low-ionic-strength (1–10 mM) buffers that do not fully represent the ionic strength or composition of saliva. It is recognized that the candidacidal activities of Hsts are closely tied to ionic strength of the incubation medium (Xu et al., 1999; Helmerhorst et al., 2001), and that selective divalent cations inhibit Hst killing (Xu et al., 1991). However, it is not known whether the masking effect of saliva by these critical ions is due to inhibition of Hst binding with target cells or disruption of later candidacidal effects. Hst 5 binding and internalization are required to initiate cytotoxic effects on yeast cells (Edgerton et al., 1998) that are characterized by efflux of small ions and ATP (Koshlukova et al., 1999). The purpose of the present studies is to identify inorganic ions of saliva that modify Hst 5 fungicidal activity and determine the point of inhibition of these interactions between Hst 5 and C. albicans.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Candidacidal Assay
Antifungal activity of Hst 5 was examined by microdilution plate assay (Koshlukova et al., 1999). Briefly, C. albicans cells were grown in YNB medium, washed twice with 10 mM sodium phosphate buffer (Na2HPO4/NaH2PO4) (pH 7.4), re-suspended (2.5 x 105 cells/mL), and incubated for 1 hr with Hst 5 (31 µM). Cells were diluted, plated onto Sabouraud Dextrose agar, and incubated for 24 hrs at 37°C. Cell survival was calculated as (number of colonies recovered from Hst 5-treated cells/colonies from control cells) x 100. All candidacidal assays were performed in triplicate. We used Student’s t test to determine statistical significance between groups.
Ionic Replacement Experiments
For candidacidal assays with ion substitutions or additions, incubation buffers were altered before the addition of Hst 5 according to the ionic species tested. Anion replacements were made in 10 mM sodium phosphate buffer supplemented with NaCl (from 5 to 100 mM), or by replacement of the Cl- counter-ion with one of the following: I-, CO3-, or gluconate. Cation replacements were made in 10 mM sodium phosphate buffer, containing CaCl2 (from 0.5 to 25 mM), or by replacement of Ca2+ with Mg2+ or SO42+. For pH experiments, sodium phosphate buffer (10 mM) was adjusted through the use of monobasic and dibasic phosphate salts to pH 5 to 9. The assay buffer at pH 4 was additionally adjusted with acetic acid. C. albicans cells were washed twice with assay buffer and maintained at the same pH throughout the assay.
Extracellular ATP Bioluminescence Assay
Measurement of extracellular ATP levels was as previously described (Koshlukova et al., 2000) with the following modifications. C. albicans (106 cells) was mixed with Hst 5 (31 µM) and incubated at 37°C for 20 min, followed by the addition of CaCl2 (5 mM or 10 mM), MgCl2 (5 mM or 10 mM), or KCl (20 mM or 50 mM). Cells were incubated for an additional 2, 5, 10, 25, and 40 min, and extracellular ATP was measured at each indicated time point. Extracellular ATP concentrations were determined from ATP standard curves for each experiment and expressed as nmoles of ATP per 106 cells.
Analysis of Hst 5 Binding to C. albicans by Flow Cytometry
We previously reported that both FITC-labeled and 125I-labeled Hst 5 have equivalent activity with the unlabeled protein (Edgerton et al., 1998; Koshlukova et al., 2000). For pre-treatment experiments, C. albicans cells were mixed with MgCl2 (10 mM), CaCl2 (10 mM), or KCl (50 mM) for 20 min, then FITC-Hst 5 (31 µM) was added and incubated for an additional 40 min at 37°C with shaking. Control cells were treated with FITC-Hst 5 (31 µM) alone. For post-treatment experiments, cells were treated with FITC-Hst 5 alone for 20 min, then were mixed with MgCl2 (10 mM), CaCl2 (10 mM), or KCl (50 mM), and incubated for an additional 40 min. Samples were diluted to a final volume of 1 mL, and 15,000 cells were analyzed on a FACSCAN flow cytometer (Becton Dickinson, San Jose, CA, USA) with a 15-mW argon laser at 488-nm excitation. Data are expressed as histograms of fluorescence (FL1) vs. counted cellular events.
Analysis of Hst 5 Binding to C. albicans with 125I-Hst 5
125I-labeled Hst 5 was prepared and binding experiments were performed as previously described (Edgerton et al., 1998). For pre-treatment experiments, C. albicans cells were mixed with CaCl2 (5 or 10 mM) for 20 min before the addition of 125I-Hst 5 (31 µM); for post-treatment experiments, cells with bound 125I-Hst 5 were washed twice in TES buffer containing 5 mM or 10 mM CaCl2 before total bound 125I-Hst 5 was measured.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Although a general trend in reduction of Hst 5 killing with increasing ionic strength was found, ion replacement in the incubation medium showed no statistically significant differences among individual anions at relevant physiological concentrations (at or below 50 mM). However, survival of Hst-5-treated cells (or inhibitory ionic effects) was statistically different (p < 0.05) between anions at high ionic concentrations (100 mM), with the following rank order: CO3- ~ gluconate > Cl- ~ I-. Analysis of these data, where replacement ions were used, shows that Hst 5 killing is not sensitive to anionic composition of the extracellular media at concentrations of relevant anions found in saliva, but is more related to overall ionic strength of the medium.
To determine whether the pH of the extracellular medium found in saliva may influence Hst 5 activity, we tested candidacidal activity of Hst 5 placed in solution at pH ranging from 4 to 9. The cytotoxicity of Hst 5 was unaffected by pH in physiological ranges from 5.0 to 7.5; however, 43 ± 3% reduction in killing was found by incubation of Hst 5 in medium with a pH 4, whereas increasing the extracellular pH to 8 or above reduced killing by 50–60%.
Effects of Extracellular Divalent Cations on Hst-5-induced Killing of C. albicans Cells
Since the major divalent cations in saliva are magnesium and calcium (Table
), we tested whether these cations could alter Hst 5 killing. The addition of 2 mM of MgCl2 provided only 22 ± 1% protection of cells from Hst-5-induced killing, while increasing the extracellular concentration of MgCl2 to 25 mM inhibited Hst 5 killing by 42 ± 2% (Fig. 1). Since this inhibition of killing was equivalent to that provided by the addition of 50 mM Cl-, experiments that substituted MgSO4 for MgCl2 in the extracellular medium were done and resulted in nearly identical Hst 5 killing. Thus, the detected inhibitory effects were due to [Mg2+], although this ion was equivalent to 50 mM Cl- in its ability to protect against Hst 5 killing.
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). Increasing extracellular [Ca2+] to 2 mM resulted in 89 ± 9% inhibition of killing, while nearly complete inhibition (95 ± 5%) of Hst 5 candidacidal effects was achieved at an extracellular concentration of 5 mM CaCl2. Since reduction in Hst 5 killing in the presence of extracellular calcium might be due to the inhibition of Hst 5 binding with C. albicans, we examined growth of C. albicans DB9 expressing intracellular Hst 5 in media supplemented with 5 mM CaCl2. In contrast to the complete inhibition of exogenous Hst-5-induced candidacidal activity in the presence of 5 mM CaCl2, shown above, no statistically significant differences in the reduction in culture growth following endogenous intracellular production of Hst 5 were found in medium containing a total of 6 mM CaCl2 over 10 hrs of induction. These results suggested that a major effect of extracellular Ca2+ is through inhibition of Hst 5 binding or uptake with C. albicans cells.
Extracellular Divalent Cations Alter Hst-5-induced ATP Efflux from C. albicans Cells
Since efflux of ATP occurs as an initial event in Hst 5 interaction with C. albicans, we measured the effects of extracellular ions on the Hst-5-induced efflux of ATP from cells. Cells pre-treated (as for ion replacement experiments) with 10 mM CaCl2 had no measurable extracellular ATP efflux, while ATP release was reduced by 55% with pre-treatment of cells with 10 mM MgCl2. We next measured alterations in ATP efflux upon the addition of CaCl2, MgCl2, or KCl following a 20-minute course of Hst-5-induced ATP release. Hst 5 treatment of cells under standard conditions initiated a steady increase in extracellular ATP (Fig. 2, solid circles). Addition of extracellular KCl (Fig. 2, open triangles, 20 mM; solid triangles, 50 mM) caused a rapid brief spike in ATP efflux. Extracellular ATP efflux then recovered to untreated levels after 40 min in 20 mM KCl and partially recovered in 50 mM KCl. Addition of MgCl2 following 20 min of incubation with Hst 5 also caused a spike and recovery of ATP efflux; however, MgCl2 (Fig. 2, open diamonds, 5 mM; solid diamonds, 10 mM) caused more total inhibition of release of ATP by 60 min than for cells incubated with 50 mM KCl. In contrast to these results, the addition of 10 mM CaCl2 resulted in an immediate loss of Hst-5-induced efflux of ATP, so that by 60 min, extracellular levels of ATP were reduced to nearly basal levels (Fig. 2, open squares, 5 mM; solid squares, 10 mM). The addition of 10 mM CaCl2 20 min after incubation with Hst 5 resulted in 80% reduction of Hst 5 killing, and was equivalent to loss of killing caused by initial pre-treatment of cells with CaCl2.
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). Thus, reduction in ATP efflux from KCl-treated cells was not a consequence of reduced Hst 5 binding with yeast. However, pre-incubation of cells with 10 mM MgCl2 substantially reduced the total amount of cell-associated Hst 5 (Fig. 3B) and was qualitatively similar to levels of Hst 5 following the post-binding addition of 10 mM MgCl2. Thus, the reduction of ATP efflux by MgCl2 is likely a consequence of loss of Hst 5 binding with cells. Pre-treatment of C. albicans cells with 10 mM CaCl2 even further reduced the amount of bound Hst 5 compared with 10 mM MgCl2, while the addition of CaCl2 following 20 min of Hst 5 incubation did not reduce the total bound protein, as assessed by FACSCAN (Fig. 3C). However, we noted that the addition of CaCl2 following pre-incubation with Hst 5 resulted in some aggregation of C. albicans cells. Therefore, FACSCAN results were qualitatively confirmed by measurement of 125I-labeled Hst 5 binding to C. albicans cells with CaCl2. Pre-treatment of cells with 5 mM or 10 mM CaCl2 reduced 125I-Hst 5 binding by 71% and 79%, respectively. Incubation of cells with 125I-labeled Hst 5 for 20 min, followed by the addition of 5 mM or 10 mM CaCl2, resulted in loss of 69% and 71% of bound Hst 5, respectively, indicating that calcium-induced aggregation indeed masked the loss of Hst 5 binding when measured by FACSCAN analysis. These results show that CaCl2 can prevent binding of Hst 5 to C. albicans cells, as well as disassociate bound Hst 5 from the cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast, the inhibitory effects of calcium and magnesium for Hst 5 killing were more pronounced than for any of the tested anions as a result of inhibition of binding of Hst 5 to C. albicans cells. The addition of CaCl2 following Hst 5 pre-incubation resulted in disassociation of 70% of previously bound Hst 5, suggesting that Ca2+ disrupts Hst 5 binding with C. albicans cells rather than associating with Hst 5 itself. This is in agreement with other reports that Hst 5 is a metalloprotein that binds zinc and copper but not other divalent ions, including calcium (Grogan et al., 2001; Gusman et al., 2001). Thus, extracellular calcium has a profound effect on Hst 5 fungicidal activity, while magnesium has a minimal inhibitory effect within the physiological concentration ranges found in saliva.
Our observations on the effects of extracellular CaCl2 in the reduction of Hst 5 activity show that Ca2+ causes rapid loss of Hst-5-induced ATP efflux yet does not affect the killing of endogenously produced Hst 5. This finding suggests the possibility that ATP release is parallel to the primary site of Hst 5 activity but is not the actual target for Hst 5. Thus, ATP release may be a marker of the effect of Hst 5 on one of two or more closely linked C. albicans proteins.
Most of the divalent ions found in whole saliva are Ca2+ and Mg2+, although the total amount of Ca2+ varies from 0.2 to 4.7 mM (Ferguson, 1989; Larsen et al., 1999). Calcium present in saliva may be in free cationic form or complexed to salivary phosphoproteins, including proline-rich proteins (Madapallimattam and Bennick, 1990), statherins (Hay and Moreno, 1989), histatin 1 (Oppenheim et al., 1986), and cystatins (Baron et al., 1999). In this regard, mean ionic free [Ca2+] in saliva from 20 individuals was reported to be 0.53 mM compared with a total calcium concentration in the same group of 1.03 mM (Matsuo and Lagerlöf, 1991). Thus, free ionic calcium available to modulate the antifungal activity of salivary histatins in vivo is likely to be highly dependent on concentrations of these salivary proteins. In addition, dietary calcium or conditions that increase free salivary calcium may have an impact on the antifungal efficacy of saliva.
Calcium is a potent inhibitor of Hst 5 candidacidal activity at physiological concentrations and may be the primary salivary ion responsible for the masking effect of saliva. This knowledge is crucial in consideration of the potential therapeutic uses of Hst 5 in the oral cavity.
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ACKNOWLEDGMENTS |
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Received November 18, 2002; Last revision April 17, 2003; Accepted May 30, 2003
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REFERENCES |
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