Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología1, and Departamento de Enfermería I2, Universidad del País Vasco, Apartado 699, E-48080 Bilbao, Vizcaya, Spain
Author for correspondence: José Pontón. Tel: +34 94 6012855. Fax: +34 94 4649266. e-mail: oipposaj{at}lg.ehu.es
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ABSTRACT |
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Keywords: Candida albicans, adhesion, salivary secretory IgA, plastic, monoclonal antibodies
Abbreviations: sIgA, secretory IgA
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INTRODUCTION |
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C. albicans can adhere to a variety of plastic materials, including the acrylic used to make dental prostheses (MacFarlane, 1990 ). Several factors are involved in the adhesion of C. albicans to plastic materials. Among them, surface hydrophobicity, electrostatic forces, presence of sucrose in the culture medium and germ tube formation seem to play a key role in the adhesion process (MacFarlane, 1990
). Tronchin et al. (1988)
and San Millán et al. (1996)
have shown that the adhesion of C. albicans to polystyrene, a model system to study the adhesion of C. albicans to plastic materials, correlates with germ tube induction, since conditions which promote filamentation increased adhesion and those inhibiting germination decreased the adhesion. The use of agerminative and filamentous mutants has confirmed this observation (Hawser & Douglas, 1994
; San Millán et al., 1996
. The increased ability of germ tubes to adhere to plastic materials has been correlated with the expression of a fibrillar layer on the surface of the cell wall which contains adhesins of >200, 200, 68 and 60 kDa (Tronchin et al., 1988
).
The adhesion of C. albicans to oral surfaces is modulated by different salivary components, including mucins (Edgerton et al., 1993 ), other salivary proteins (Cannon et al., 1995
) and secretory IgA (sIgA). The effect of these components on the adhesion of C. albicans to oral surfaces may be very different since, while some of them can enhance adhesion, others can bring about inhibition (Epstein et al., 1982
; Vudhichamnong et al., 1982
; Edgerton et al., 1993
). Inhibition of adhesion of C. albicans to oral surfaces is thought to be an important strategy in preventing oral candidiasis. Various approaches have been resorted to in an attempt to inhibit C. albicans adhesion to host cells or to plastic surfaces. Such approaches include the use of C. albicans antigens, host proteins, antifungal agents and antibodies (Epstein et al., 1982
; Critchley & Douglas, 1987
; Vuddhhakul et al., 1988
; Edgerton et al., 1993
). Bendel et al. (1993)
demonstrated inhibition of adhesion of C. albicans to buccal epithelial cells with an mAb against the ß2-integrin subunit
M, and Miyakawa et al. (1992)
demonstrated reduced adherence of C. albicans to buccal epithelial cells using an mAb against factor 6, an epitope in the cell wall mannan responsible for serotype A specificity in C. albicans. We have previously shown that adhesion of C. albicans to polystyrene can be partially inhibited with an mAb directed against an epitope of antigen 6 expressed on the >200 kDa component of the fibrillar adhesins of C. albicans (San Millán et al., 1996
). In this report, we have studied the role of whole saliva in the adhesion of C. albicans to polystyrene and we have used three mAbs directed against epitopes of cell wall mannoproteins to mimic the inhibitory effect observed with salivary sIgA on the adhesion of C. albicans to polystyrene.
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METHODS |
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Saliva and mAbs.
The experimental protocols were approved by the Institutional Review Board of the School of Medicine and Odontology at the University of Basque Country and all the subjects gave informed consent to participate. Unstimulated whole saliva from four healthy C. albicans carriers and non-carriers was collected. In every case, the saliva was centrifuged at 6000 g for 30 min at 4 °C and the supernatant was stored at -80 °C until used. The salivas were studied by both indirect immunofluorescence and spectrofluorometry assays against three C. albicans oral isolates (UPV-94392, UPV-94393 and UPV-94394). The pattern of reactivity of each saliva was the same with each strain so the most reactive salivas from three asymptomatic C. albicans oral carriers were used. The concentration of IgA in saliva was estimated by ELISA using purified human IgA (Sigma) as a control.
In one experiment, whole saliva was adsorbed with formalin-killed germ tubes or yeast cells of C. albicans NCPF 3153 suspended at 1010 cells (ml PBS)-1. Adsorption was performed by mixing equal volumes of the adsorbing suspension and saliva. After incubation in a rotatory mixer for 2 h at room temperature, the organisms were removed by centrifugation. Supernatants were concentrated to the initial volume and stored at -80 °C until used. The C. albicans cells used in the adsorption of saliva were washed with PBS (7 mM Na2HPO4, 2·7 mM NaH2PO4 and 145 mM NaCl; pH 7·2) and components attached to the cell wall surface of C. albicans were then released by treatment with 2·5 M NaI in PBS for 1 h at room temperature. Cells were removed by centrifugation, and the supernatant was dialysed against PBS for 48 h at 4 °C. Eluted components were concentrated to the initial volume with polyethylene glycol 20000 and stored at -80 °C until used. The eluted components were analysed by SDS-PAGE as previously described (Pontón et al., 1996 ). The total amount of protein loaded per lane was 5 µg.
To obtain saliva depleted of IgA, saliva samples were applied to an Affi-Gel 10 column (Bio-Rad) coupled to an anti-human IgA (Sigma). The saliva depleted of IgA was concentrated to the initial volume as described above and the depletion of IgA was assessed by SDS-PAGE and Western blotting with both a goat anti-human IgA and a goat anti-human secretory component (Pontón et al., 1996 ). The purity of the IgA eluted from the affinity chromatography column was also checked by SDS-PAGE and Western blotting. In some experiments, an irrelevant IgA purified from human colostrum (Sigma) was used as a control. Two IgM mAbs (C7 and B9E) and one IgA (N3B) were produced following standard methods with splenocytes from BALB/c mice immunized by subcutaneous injections of a partially purified antigen of 260 kDa from a germ tube extract (B9E and N3B) (Pontón et al., 1993
) or a cell wall stress mannoprotein of 200 kDa (C7) (Pontón et al., 1996
). mAbs were purified from ascites fluid by affinity chromatography on an anti-mouse IgM or an anti-mouse IgA (Sigma) coupled to an Affi-Gel 10 column, essentially as described for purifying sIgA. Purified antibodies were dialysed against PBS before use. Their reactivity against cell wall extracts from C. albicans was assessed by SDS-PAGE and Western blotting as previously described (Pontón et al., 1993
). The reactivity of mAb C7 was studied in DTT extracts after mild periodate oxidation [20 min at room temperature with 10 mM sodium metaperiodate (Sigma) in 100 mM acetate buffer, pH 5·3].
Adherence assays.
Two adhesion assays were used. The visual assay was performed as previously described (San Millán et al., 1996 ). To determine the effect of the saliva on adherence, the yeast cells were inoculated in medium 199 containing 520 µl whole saliva or sIgA-depleted saliva. In one experiment, the cells were grown in an Erlenmeyer flask containing medium 199. Germ tubes were induced at 37 °C with shaking and yeast cells were grown at 25 °C. After 40 and 140 min growth, the cells were washed with medium 199 at 4 °C, counted in a haemacytometer and transferred to polystyrene Petri dishes at a final concentration of 5x106 cells ml-1 medium 199 or a mixture of medium 199 and saliva. The Petri dishes were incubated at 25 °C for 40 min and the adhesion was quantified as described above. The controls consisted of cells incubated in Petri dishes for 40 and 140 min at both 37 and 25 °C.
In the other assay, adhesion was quantified spectrofluorometrically. Yeast cells were inoculated in 350 µl medium 199, pH 6·7, at a final concentration of 1x106 cells ml-1 and incubated at 37 °C in 24-well tissue culture polystyrene plates. After washing, the plates were stained with 0·1% calcofluor white in saline for 20 min at room temperature. Plates were washed again and the fluorescence was read in a spectrofluorometer (Fluoroskan Ascent; Labsystems) with an excitation filter of 358 nm and an emission filter of 460 nm. The controls used to examine possible interference from autofluorescence and non-specific binding consisted of wells without adherent cells and their background fluorescence was subtracted from that shown by the test wells. All values quoted represent mean figures derived from at least four independent assays. To determine the effect on adherence of the saliva, the purified secretory IgA and the mAbs, the yeast cells were inoculated in medium 199 containing 280 µl saliva or 20 µg of the purified sIgA or the mAb. In experiments where a combination of two or three mAbs were studied, 10 µg or 6·66 µg of each mAb was used, respectively.
Statistics.
The ANOVA test was used to assess the significance of differences between means in adherence assays. Data were considered significant at P< 0·05.
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RESULTS |
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Influence of whole saliva on the adhesion of C. albicans to polystyrene
The kinetics of germination and adhesion to polystyrene of C. albicans 3153 was initially studied at 37 °C by the visual method in the absence of saliva (Fig. 1). Under these conditions, 20% of C. albicans 3153 cells showed short germ tubes (with a length approximately one or two times greater than that of the mother yeast) after 40 min incubation. Longer periods of incubation resulted in an increase in the percentage of germination and an extension in the length of the germ tubes. The maximum level of germination (96·8%) was reached at 140 min. At this time, the length of the germ tubes was approximately 10 times greater than that of the mother yeast. Adhesion of C. albicans 3153 to polystyrene increased with germination and it also reached a plateau (76·7%) at 100 min. In the presence of whole saliva, the germination of C. albicans 3153 was very similar to the control without saliva and no statistically significant differences were observed when germination in the presence and absence of whole saliva was compared (Fig. 1
). In contrast to the adhesion to polystyrene in the absence of saliva, the presence of whole saliva enhanced the adhesion to polystyrene (434·7%) of C. albicans 3153 cells grown for 40 min at 37 °C but decreased the adhesion of germinated cells by 68·6% at 140 min incubation.
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Influence of sIgA-depleted saliva on the adhesion of C. albicans to polystyrene
The results obtained suggested that one or more components present in whole saliva was inhibiting the adhesion of C. albicans 3153 germ tubes to polystyrene. Since sIgA is one of the salivary components which are believed to be involved in the inhibition of the adhesion of micro-organisms to oral surfaces we depleted the saliva of sIgA and studied the ability of the sIgA-depleted saliva to inhibit the adhesion of C. albicans 3153 to polystyrene. As shown in Fig. 2, C. albicans 3153 germinated in the presence of sIgA-depleted saliva with similar kinetics to that shown by the cells in the absence of saliva. However, the presence of sIgA-depleted saliva in the adhesion experiments enhanced the adhesion of C. albicans 3153 to polystyrene when compared to the adhesion of the cells in the absence of saliva, especially during the first 80 min of incubation. Adhesion and germination of C. albicans 3153 were not affected by the presence of IgA purified from human colostrum.
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DISCUSSION |
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Although C. albicans can adhere directly to plastic materials (Tronchin et al., 1988 ; Kennedy et al., 1989
; San Millán et al., 1996
), prostheses introduced into the oral cavity are rapidly coated by a pellicle containing selectively adsorbed salivary components, including high-molecular-mass mucin, amylase and sIgA (Edgerton & Levine, 1992
) which can have opposite effects on the adhesion of C. albicans. Preincubation of acrylic strips with whole saliva has been shown to decrease adhesion of C. albicans to denture acrylic in vitro (Samaranayake et al., 1980
; McCourtie & Douglas, 1981
). In contrast, coating denture acrylic with whole saliva as well as parotid and submandibular-sublingual saliva enhanced Candida adhesion (Vasilas et al., 1992
). The results presented in this study confirm both effects of saliva on the adhesion of C. albicans to plastic materials, since whole saliva decreased or enhanced the adhesion of C. albicans to polystyrene depending on the morphological phase of C. albicans. Whole saliva decreased adhesion of hyphae of C. albicans 3153 to polystyrene while the adhesion of yeast cells was enhanced. The enhancement of adhesion of blastospores to polystyrene promoted by whole saliva was confirmed with the mutant CA2, which was derived from C. albicans 3153. The adhesion of this mutant unable to form germ tubes was very low in the absence of whole saliva but its adhesion to polystyrene was significantly enhanced in the presence of whole saliva from different donors. Different components of the salivary pellicle may be responsible for the enhancement of adhesion observed in the yeast cells. Edgerton et al. (1993)
have shown that mucin-containing human submandibular-sublingual saliva is capable of significant enhancement of adhesion of yeasts cells. However, other components, including antibodies, may also enhance the adhesion of yeast cells to polystyrene. In fact, we have described an mAb directed against the C. albicans adhesins involved in attachment to plastic materials which enhanced the adhesion of yeast cells to polystyrene (San Millán et al., 1996
).
Although other salivary components may also be involved, the decrease in adhesion of germ tubes to polystyrene seemed to be related to the presence in whole saliva of sIgA, since the inhibitory effect was not observed in sIgA-depleted saliva. Interestingly, Umazume et al. (1995) have shown that inhibition of adhesion of C. albicans to epithelial tumour cells caused by whole saliva was almost completely abolished by an anti-sIgA antibody, and saliva from patients receiving chemotherapy for oral carcinoma, which had reduced concentrations of sIgA, showed reduced inhibition of C. albicans adhesion. The inhibitory effect mediated by sIgA is not related to an inhibition of germ tube induction, since similar levels of germination were observed in C. albicans cells in the absence or presence of sIgA. The effect is likely to be produced by blocking the adhesins present on the cell wall surface of the fungus, since adsorption of the saliva with yeast cells or germ tubes, a procedure which removed components bound to the fungal surface, reduced the inhibitory effect of whole saliva. Conversely, the salivary components eluted from the surface of the fungal cells showed an inhibition of adhesion similar to that observed with untreated whole saliva.
Inhibition of adhesion of C. albicans to polystyrene by mAbs
Knowledge of the mechanisms for attachment of C. albicans to plastic materials has allowed for the development of strategies to block the adhesion of the fungus. Different approaches have been tried to block the adhesion of C. albicans to plastic materials, including the use of sugars (Edgerton et al., 1993 ), histatin 5 (Edgerton et al., 1995
) and saliva (Samaranayake et al., 1980
; McCourtie & Douglas, 1981
; McCourtie et al., 1986
). Since the adhesion of C. albicans to plastic has been shown to be mediated by cell wall mannoproteins (Tronchin et al., 1988
) and salivary sIgA reacts with mannoproteins of C. albicans of different molecular masses (Pontón et al., 1996
), in this study we have tried to mimic the inhibitory effect on C. albicans adhesion mediated by sIgA with mAbs directed against epitopes expressed on cell wall surface antigens of C. albicans (Pontón et al., 1993
). All mAbs studied inhibited the adherence of C. albicans to polystyrene at levels equivalent to those for purified sIgA. Interestingly, the highest reduction in the adhesion was obtained with mAb N3B, an IgA which shows a reactivity with mannoproteins of C. albicans reminiscent of that shown by salivary sIgA. However, the results obtained with purified sIgA and mAbs are not directly comparable with those obtained with whole saliva since purified antibodies were used at a concentration 30 times higher than that of the sIgA in saliva. These results extend our previous observation that mAbs directed against epitopes expressed on C. albicans adhesins involved in attachment to polystyrene can inhibit adherence of C. albicans to this plastic (San Millán et al., 1996
). It is worth noting that the inhibitory effect can be enhanced by combining mAbs directed against different epitopes. It seems likely that mAbs mimic the inhibitory effect of saliva on the adherence of C. albicans to polystyrene through a different mechanism, since mAb B9E inhibited germination of C. albicans (San Millán et al., 1996
) while, as demonstrated in this study, sIgA did not. Historically, there has been controversy over the relative importance of antibody- and cell-mediated protection in candidiasis (Casadevall, 1995
; Cassone et al., 1997
; Casadevall et al., 1998
). Data presented in this paper provide further support to the notion that antibodies, and sIgA in particular, can modify the course of C. albicans infections associated with plastic devices by modulating the adhesion of the fungus to these materials.
In conclusion, we have demonstrated that whole saliva has a dual role in the adhesion of C. albicans to polystyrene, since it can enhance or inhibit the adhesion depending on the morphological phase of the fungus. The inhibitory effect shown by salivary sIgA can be mimicked by mAbs directed against epitopes of cell wall mannoproteins. If this effect is also observed in the adhesion of C. albicans to acrylic, the use of mAbs may open a new approach for inhibiting the adhesion of C. albicans to dental prostheses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 December 1999;
revised 1 June 2000;
accepted 16 June 2000.