Effect of antifungal agents on the binding of Candida albicans to immobilized amino acids and bovine serum albumin

Khalid Islam* and Stephen P. Hawser

Lepetit Research Center, Via R. Lepetit 34, 21040 Gerenzano (VA), Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study the effects of different antifungal agents on the binding of Candida albicans yeast cells to different supports were examined. Pre-treatment with amphotericin B or dithiothreitol (DTT) severely reduced the ability of C. albicans yeasts to bind to plastic, while the effects of pre-treatment with fluconazole, ketoconazole or flucytosine were less marked. Both DTT and amphotericin B reduced the binding of yeasts to bovine serum albumin (BSA) and amino acids at low concentrations, while the other antifungal agents were effective at concentrations several-fold higher than their MICs. These data suggest that DTT and amphotericin B affect the yeast cell wall components, and alter both hydrophobic interactions with plastic, and the more specific interactions with BSA and amino acids. By contrast, the effect of the azoles and flucytosine appears to be largely restricted to hydrophobic interactions.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A recent study from this laboratory, 1 using the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5- carboxanilide inner salt (XTT) tetrazolium salt assay and light microscopy, demonstrated that it is possible to assess the binding of Candida albicans to different surfaces. It was observed that C. albicans binds efficiently to plastic, immobilized bovine serum albumin (BSA) and to the L-configurations of alanine, arginine, leucine, lysine and proline but not to the D-configurations or to any other of the natural amino acids. 1

The pathogenicity of C. albicans appears to be related, in part, to its ability to attach to different types of host surface. The adhesion process is highly dependent upon the nature of the candida cell surface and that of the substratum. 2 3 4 There is sparse information concerning the effects of antifungal agents on the ability of C. albicans to bind to different substrates. Some reports have suggested that certain agents, at sub-MIC concentrations, may alter the cell surface hydrophobicity of yeasts, and may also affect the ability of yeasts to bind to different human cell types, and several components of the extracellular matrix (ECM). 5 6 7 In this study, the effects of various concentrations of antifungal agent on the ability of yeasts to bind to plastic, immobilized BSA and to immobilized amino acids, was investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antifungal agents, chemicals and reagents

Amphotericin B, flucytosine, Type B gelatin from bovine skin, type IV BSA, phenazine methosulphate, XTT tetrazolium salt and dithiothreitol (DTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). L-Alanine, L-arginine, L-leucine, L-lysine and L-proline were purchased from Calbiochem (La Jolla, CA, USA). Fluconazole was obtained from Pfizer (Sandwich, UK) and ketoconazole from Janssen Pharmaceutica (Beerse, Belgium).

Organisms, culture conditions and binding assay

C. albicans strains ATCC 10231 and 90028 were obtained from the American Type Culture Collection; CA444, CA74 and CA6406 were kindly provided by Prof. J.-P. Latge (Paris, France), Professor M. Monod (Laussane, Switzerland) and Professor D. Kerridge (Cambridge, UK), respectively. C. albicans strains were grown and prepared for use in binding experiments as described previously. 8

The binding of C. albicans to the different supports was performed as described previously. 1 The effects of pre-treatment with antifungal agents or DTT were examined after incubation of yeasts with an antifungal agent at the MIC, or with 10 mM DTT for 4 h at 25°C, with shaking. Free antifungal agent or DTT was then removed by three washes in phosphate buffered saline (PBS). Following washing, 100 µL of yeast suspension in PBS (1 x 10 5 C. albicans cells/mL) was allowed to bind to plastic, immobilized BSA or immobilized amino acids as previously described. In concentration-dependent assays, yeasts were pre-incubated in solutions of 0.001- 100 mM DTT, 0.001- 1.0 mg/L amphotericin B, fluconazole and ketoconazole or 0.01- 10 mg/L flucytosine for 4 h at 25°C with shaking. The cells were then washed three times with PBS to remove free antifungal agent or DTT. Following washing, 100 µL aliquots of yeasts in PBS (1 x 10 5 C. albicans cells/mL) were allowed to bind to the various surfaces, and binding was quantified using the XTT tetrazolium assay and routinely confirmed using light microscopy. 1

Effects of pre-incubation with DTT or antifungal agents on yeast viability

The effects of pre-incubation of yeasts with different concentrations of DTT or antifungal agent for 4 h were determined. Yeasts were pre-incubated as described above, free DTT or antifungal agent removed by washing three times with PBS, and samples from each pre-incubation or from untreated yeasts were removed and plated on to Sabouraud dextrose agar (Difco). The plates were incubated for 24 h at 25°C and the number of cfu determined. The cfu from pre-treatment experiments were expressed as a percentage of the cfu from untreated control cells.

MIC determinations

MIC determinations were performed using a microtitre plate format as described previously 8. Briefly, wells containing 1.5 x 10 3, 1.5 x 10 4 or 1 x 10 5 cfu in RPMI-1640 medium buffered with 0.15 M 3-[N-morpholino]propanesulphonic acid (MOPS) were used. To appropriate wells, antifungal agents were added to give final concentrations of 0.003- 128 mg/L. The plates were incubated for 48 h at 35°C and the MIC read as the lowest concentration of antifungal agent which resulted in no discernible growth of the cells.

Statistical analysis

Data from binding experiments were compared by Student's t-test using the StatWorks program. Significant differences between data were expressed at P < 0.01, P < 0.05 or P < 0.001.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Five different C. albicans strains were used in preliminary studies; these comprised three clinical isolates (CA74, CA444 and CA6406) and ATCC 10231 and ATCC 90028. As all strains behaved similarly, the ATCC 10231 strain was chosen for all further studies.

MIC determinations and effects of pre-treatment with antifungal agent or DTT on cell viability

The MICs of amphotericin B, fluconazole, ketoconazole and flucytosine for C. albicans ATCC 10231 were 0.06, 0.03, 0.06 and 0.13 mg/L, respectively. Similar MICs were obtained for each antifungal agent when tested against inocula of 1.5 x 10 3, 1.5 x 10 4, or 1 x 10 5 cfu/well (data not shown), suggesting that the activity of each agent was not affected by the inoculum size.

Pre-treatment of yeast cells at sub-inhibitory concentrations or at the MIC for 4 h did not reduce the viability of yeast cells. However, when the cells were pre-treated with 1 mg/L of amphotericin B, fluconazole, ketoconazole or 10 mg/L of flucytosine, the number of cfu on agar was reduced by 76%, 15%, 18% and <3%, respectively, in comparison with the untreated control cells. Pre-treatment with DTT typically did not cause reductions in viability, though the viability of cells pre-treated in the presence of 100 mM DTT for 4 h was reduced by about 6%.

Effects of pre-treatment of yeasts with DTT or antifungal agents on binding

Pre-treatment of yeasts with 1 g/L BSA did not affect the ability of yeasts to bind to the non-coated (plastic) surface. However, pre-treatment with either 10 mM DTT or 1 x MIC of amphotericin B significantly reduced (P < 0.001) the binding by about 85%. By contrast, pre-treatment with the other antifungal agents caused reductions in binding to plastic of only about 40- 50% (P < 0.01) (Figure 1.)



View larger version (36K):
[in this window]
[in a new window]
 
Figure 1. The effects of pre-treatment with BSA, DTT or with amphotericin B, fluconazole, ketoconazole or flucytosine on the binding of yeast-phase C. albicans to plastic (a) and to immobilized BSA (b). Cells were pre-treated with 1 g/L BSA, 10 mM DTT or 1 x MIC of each antifungal agent for 4 h. Thereafter, the cells were washed to remove BSA, DTT or antifungal agent in solution. Binding to plastic was quantified using the XTT tetrazolium salt assay. The bars represent the mean ± S.D. (n = 21) from a typical experiment.

 
The effects of pre-treatment of yeasts with BSA, DTT or antifungal agents on the ability of cells to bind to immobilized BSA were also examined. Figure 1 shows that pre-treatment of yeasts with 1 g/L BSA significantly (P < 0.001) reduced the binding of yeasts to immobilized BSA; see also Hawser & Islam 1 Similarly, pre-treatment of yeasts with either 10 mM DTT or 1 x MIC amphotericin B also significantly (P < 0.001) reduced the binding of yeasts to the immobilized substrate (Figure 1.) Pre-treatment with other antifungal agents at the MIC did not significantly (P < 0.1) reduce the ability of yeasts to bind to immobilized BSA. For example, pre-treatment of yeasts with fluconazole, ketoconazole or flucytosine reduced the binding to BSA by 23%, 10% and 6%, respectively.

The effects of various antifungal agents or DTT on the ability of yeasts to bind to immobilized amino acids was examined. In these assays, DTT was shown to reduce the binding of yeasts to all amino acids, even at low concentrations, and complete inhibition of binding was typically observed when yeasts were pre-treated with 1 mM DTT (Figure 2.) From the dose- response inhibition curves, DTT exhibited IC 50s (concentrations inhibiting binding by 50%) of 0.02- 0.3 mM for binding of yeasts to the different amino acids (Figure 2.) Amphotericin B exhibited IC 50s of 0.01- 0.08 mg/L for all amino acids, with complete loss of binding observed at 1.0 mg/L (Figure 2.)



View larger version (22K):
[in this window]
[in a new window]
 
Figure 2. The effects of pre-treatment with DTT or antifungal agents on the binding of synchronized yeast-phase C. albicans to immobilized L-arginine ({blacksquare}), L-alanine ({circ}), L-leucine (•), L-lysine ({blacklozenge}) or L-proline ({square}). Yeasts were pre-treated with 0.001- 100 mM DTT (a), 0.001- 1.0 mg/L of amphotericin B (b), 0.001- 1.0 mg/L of fluconazole (c), 0.001- 1.0 mg/L of ketoconazole (d) or 0.01- 10 mg/L of flucytosine (e) for 4 h. Cells were then washed to remove remaining DTT or antifungal agent, and the binding of yeasts to each immobilized amino acid was quantified using the XTT tetrazolium salt assay. The values represent the mean (n = 8) and are expressed as a percentage of the binding of untreated control yeasts to the immobilized amino acid. The S.E.M. was below ±10% in these experiments.

 
The effects of fluconazole and ketoconazole on the binding of yeasts to immobilized amino acids were different from those observed with DTT or amphotericin B. For example, pre-treatment with fluconazole or ketoconazole gave IC 50 values of 0.1- 0.7 mg/L for inhibition of binding to the amino acids. Complete inhibition of binding was not observed even at high concentrations (Figure 2.) Since flucytosine has a relatively high MIC, the effects of this agent were assessed at concentrations of 0.01- 10 mg/L. The inhibition curves for flucytosine suggest that this agent affects the binding of yeasts to immobilized amino acids to a similar degree to that observed for fluconazole and ketoconazole, albeit at higher concentrations (Figure 2.) Surprisingly, pre-treatment with flucytosine at 1 mg/L reduced the binding to L-proline by about 80%, whereas binding to other amino acids was not markedly affected at this concentration (Figure 2.)


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Determination of the effects of antifungal or DTT treatments on the ability of yeasts to bind to different substrates showed that at the MIC of amphotericin B, or with 10 mM DTT the binding of yeasts to plastic or to immobilized BSA was drastically reduced. The action of amphotericin B or DTT on binding could be a consequence of either (i) a reduction in cell viability or (ii) direct action on the binding of yeasts to a support. However, no appreciable change in the cfu counts was observed between the pre-treated or untreated cells, except at high concentrations. These agents, therefore, probably act directly on the binding of yeasts to the support. Binding of yeasts to plastic has been suggested to be related to the hydrophobicity of yeast cells 2, 9 whereas binding to BSA is more likely to involve ligand- protein or protein- protein interactions.

By contrast, the azole agents and flucytosine, at the MIC, only partially affected the binding of yeasts to plastic, and minimally affected the binding to immobilized BSA. In terms of the binding of azole-treated yeasts to plastic, the observed reductions in binding are most likely to be due to changes in the cell surface hydrophobicity of yeasts. A recent report suggests that sub-MIC concentrations of fluconazole may increase the hydrophobicity of yeasts, and as a consequence, reduce the binding of yeasts to plastic and to ECM components, including fibronectin and laminin. 9 These agents do not significantly affect the binding of yeasts to BSA. These data would further suggest that binding to BSA requires more specific ligand- protein or protein- protein interactions.

The yeast cell- amino acid binding interactions are selective and stereospecific. Pre-treatment of yeasts with DTT and amphotericin B attenuated the binding of yeasts to the immobilized amino acids in a dose-dependent manner (IC 50s of 0.02- 0.3 mM for DTT and IC 50s in the sub-MIC or 1 x MIC range for amphotericin B). These agents therefore affect not only the hydrophobic interactions, for example with plastic, but also the more specific interactions such as those with BSA and amino acids, at concentrations where no effect on cell viability was observed. By contrast, the azoles and flucytosine only affected binding to amino acids at high concentrations, which also resulted in small reductions in cell viability. The observations suggest more generalized effects on the cell, which may be responsible for the observed reduction in binding to immobilized amino acids. Therefore, the azoles and flucytosine cause alterations in hydrophobicity resulting in loss of binding to plastic, albeit to a lesser extent when compared with amphotericin B or DTT, but do not affect the protein- protein or ligand- protein type interactions as observed with amphotericin B or DTT.

The binding of candida to large proteins, such as BSA and components of the ECM, is likely to involve particular amino acids. Several reports suggest that the binding of yeasts, treated with sub-inhibitory concentrations of antifungal agent, to various cell types can be attenuated. 6 , 7 , 10 It would have been expected, therefore, that pre-treatment of yeast cells with all antifungal agents would have resulted in attenuation of binding to immobilized amino acids. Only amphotericin B appears to be able to attenuate binding of yeasts when used at sub-MIC concentrations. This finding may be attributed to the mechanism of action of amphotericin B, 11 an agent which rapidly disrupts membrane integrity and is known to cause changes in the mannan content of the cell wall. 12 By contrast, the azoles exert their antifungal action by inhibition of cytochrome P 450 demethylase, 11 and flucytosine by inhibition of RNA and DNA syntheses, 11 and may require much longer pre-treatment periods in order to exert their action. 7

In conclusion, we have shown that marked differences exist in the manner in which C. albicans adheres to different supports. As adherence is a primary step in the pathogenesis of infection, a better understanding of the mechanisms of adherence of C. albicans may prove useful for future antifungal therapy. In this context, studies with amino acids, rather than plastic, mucosal surfaces, cell lines or proteins may offer advantages, and be a better analytical tool for understanding microbial interactions; for example, flucytosine-treated yeasts appeared to bind much less avidly to immobilized proline than to all the other amino acids.


    Notes
 
* Correspondence address. Hoechst Marion Roussel, Batiment Fleming Sr-5239, 102 Route de Noisy, F-93235 Romainville, Cedex, France. Tel: +33-1-4991-3259; Fax: +33-1-4991-3116; E-mail: Khalid. Islam{at}hmrag.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Hawser, S. P. & Islam, K. (1998). Binding of Candida albicans to immobilized amino acids and bovine serum albumin. Infection and Immunity 66, 140–4.[Abstract/Free Full Text]

2 . Hostetter, M. K. (1994). Adhesions and ligands involved in the interaction of Candida spp. with epithelial and endothelial surfaces. Clinical Microbiology Reviews 7,29 –42.[Abstract]

3 . Klotz, S. A., Rutten, M. J., Smith, R. L., Babcock, S. R. & Cunningham, M. D. (1993). Adherence of Candida albicans to immobilized extracellular matrix proteins is mediated by calcium-dependent surface glycoproteins. Microbial Pathogenesis 14, 133–47.[ISI][Medline]

4 . Calderone, R. A. & Braun, P. C. (1991). Adherence and receptor relationships of Candida albicans. Microbiology Reviews 55, 1–20.

5 . Hostetter, M. K. (1996). An integrin-like protein in Candida albicans : implications for pathogenesis. Trends in Microbiology 4, 242–6.[ISI][Medline]

6 . Sobel, J. D. & Obedeanu, N. (1983). Effects of subinhibitory concentrations of ketoconazole on in-vitro adherence of Candida albicans to vaginal epithelial cells. European Journal of Clinical Microbiology 2, 445–52.[ISI][Medline]

7 . Vuddhakul, V., McCormack, J. G., Kim Seow, W., Smith, S. E. & Thong, Y. H.(1988 ). Inhibition of adherence of Candida albicans by conventional and experimental antifungal drugs. Journal of Antimicrobial Chemotherapy 21, 755–63.[Abstract]

8 . Hawser, S. P. & Islam, K. (1996). Spectrophotometric determination of the morphogenetic transformation by synchronous Candida albicans : effects of antifungal agents. Journal of Antimicrobial Chemotherapy 38, 67–73.[Abstract]

9 . Hazen, K. C. & Wu, G. G. (1997). Effect of subinhibitory concentrations of fluconazole and amphotericin B on surface hydrophobicity of Candida albicans and Candida tropicalis. In Abstracts of the Ninety-Seventh General Meeting of the American Society for Microbiology, Miami, FL, 1997. Abstract F-80, p. 273. American Society for Microbiology, Washington, DC.

10 . Mehentee, J. F. & Hay, R. J. (1990). Effect of antifungal agents on the adherence of Candida albicans to murine gastrointestinal mucosal surfaces. Journal of Antimicrobial Chemotherapy 25, 111–9.[Abstract]

11 . Kerridge, D. (1988). Mode of action of clinically important antifungal drugs. Advances in Microbial Physiology 27, 1–34.

12 . Al-Bassam, T., Poulain, D., Giummelly, P., Lematre, J. & Bonaly, R. (1985). Chemical and antigenic alterations of Candida albicans cell walls related to the action of amphotericin B sub-inhibitory doses. Journal of Antimicrobial Chemotherapy 15, 263–9.[Abstract]

Received 20 April 1998; returned 17 June 1998; revised 31 July 1998; accepted 8 December 1998





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Islam, K.
Articles by Hawser, S. P.
PubMed
PubMed Citation
Articles by Islam, K.
Articles by Hawser, S. P.