Department of Oral Sciences and Orthodontics, University of Otago, PO Box 647, Dunedin, New Zealand1
Author for correspondence: Justin M. OSullivan. Tel: +44 1227 764 000. Fax: +44 1227 763 912. e-mail: jmo{at}ukc.ac.uk
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ABSTRACT |
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Keywords: co-adhesion, Candida albicans, streptococci, proline-rich proteins
Abbreviations: HA, hydroxylapatite; PRPs, proline-rich proteins; aPRPs, acidic proline-rich proteins; bPRPs, basic proline-rich proteins
a Present address: Research School of Biosciences, University of Kent at Canterbury, Canterbury CT2 7NJ, UK.
b Present address: Department of Oral and Dental Science, University of Bristol, Bristol, UK.
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INTRODUCTION |
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The oral cavity comprises diverse micro-environments containing a range of surfaces to which microbial cells can adhere, including the teeth, epithelial mucosa and the nascent surfaces created as micro-organisms bind to existing biofilms (Whittaker et al., 1996 ). All these surfaces are coated with salivary glycoproteins that are selectively bound (Douglas & Russell, 1984
; Scannapieco et al., 1989
; Nikawa & Hamada, 1990
; Edgerton et al., 1993
; Newman et al., 1993
; Rudney et al., 1995
). Co-adhesion (the binding of planktonic microbial cells to surface-bound microbial cells) is an important mechanism in microbial cell growth and survival in the oral cavity, and may be modulated by salivary molecules.
C. albicans has been isolated from dentures (Arendorf & Walker, 1979 ; Wright et al., 1985
), dental plaque (Hodson & Craig, 1972
; Arendorf & Walker, 1980
) and from sites of periodontal disease (Slots et al., 1988
; Rams & Slots, 1991
). Furthermore, adhesion of C. albicans to oral surfaces including buccal epithelial cells and acrylic (Verran & Motteram, 1987
; Branting et al., 1989
) has been shown to be influenced by oral bacteria. Therefore, there is good in vivo and in vitro evidence for Candida interactions with bacterial biofilms. Holmes et al. (1995b
) demonstrated that C. albicans cells co-aggregate with, and co-adhere to, certain species of oral streptococci, and for some species, e.g. Streptococcus gordonii, salivary proteins enhance the interactions (Holmes et al., 1995a
). Salivary-protein-enhanced co-adhesion of oral bacteria has been frequently described (see Jenkinson & Lamont, 1997
) and the saliva-enhanced binding of Streptococcus mutans to Actinomyces naeslundii, Streptococcus sanguinis and Streptococcus mitis is believed to be a significant factor in the development of early plaque (Lamont & Rosan, 1990
). However, few studies have identified the salivary components that enhance inter-microbial adhesion, or indeed that may inhibit co-adhesion (Stinson et al., 1992
).
Several studies (Gibbons & Hay, 1988 ; Gibbons et al., 1991
) have demonstrated that the acidic proline-rich proteins (aPRPs) can promote the adherence of A. naeslundii and S. gordonii cells to apatitic surfaces. These studies concluded that the acidic PRP-1 protein was cryptic in nature due to the fact that it did not bind to the cell surface of A. naeslundii LY7 cells, but rather is only recognized as a receptor for A. naeslundii LY7 and S. gordonii Blackburn cells when immobilized onto an apatitic surface (Gibbons & Hay, 1988
; Gibbons et al., 1991
). However, subsequent studies have shown that oral streptococci, when incubated with parotid saliva, adsorb salivary protein components including the aPRPs and some basic PRPs (bPRPs IB-1 to IB-9) onto their surfaces in a species- and strain-specific manner (Newman et al., 1993
). Therefore, it is possible that some streptococci are able to recognize alternate forms of PRPs, either soluble or bound. This is analogous to the suggestion that streptococci are able to bind and differentiate between soluble and immobilized forms of salivary agglutinin (Jenkinson & Demuth, 1997
) and allows bacterial cell adhesion to salivary pellicles despite the excess soluble forms of the receptor in saliva.
Since we have previously identified the bPRP IB-6 and three other PRPs as possible pellicle receptors for C. albicans ATCC 10261 adhesion (OSullivan et al., 1997 ), we investigated the hypothesis that selective adsorption of bPRPs by streptococci may enhance C. albicans adhesion within oral biofilms.
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METHODS |
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Collection and fractionation of parotid saliva.
Stimulated parotid saliva samples were collected from six donors using a modified CarlsonCrittenden device (Shannon et al., 1962 ) and stored on ice. The following proteinase inhibitors were added to the saliva samples (final concentrations): PMSF (1 mM); bisulphite (10 mM); benzamidine/HCl (2 mM); pepstatin A (2·9 µM); and aprotinin (0·3 µM). Samples were then pooled and clarified by centrifugation (12000 g, 4 °C, 15 min). For fractionation, saliva (60 ml) was freeze-dried, suspended in water and applied to a Sephacryl S200 column (XK 26/100; Pharmacia). Fractions were collected and characterized as previously described (OSullivan et al., 1997
).
Adhesion of C. albicans to saliva-coated hydroxylapatite (HA) beads.
C. albicans cells were radioactively labelled with [35S]methionine [New England Nuclear; 4·3x1013 Bqmmol-1 (1175 Ci mmol-1)] to a specific radioactivity of 0·036±0·017 c.p.m. per cell as described previously (OSullivan et al., 1997 ). Labelled cells were washed and suspended in KCl buffer (2 mM KH2PO4, 2 mM K2HPO4, 5 mM KCl, 1 mM CaCl2, pH 6·5) and numbers of C. albicans cells adhering to parotid-saliva-coated HA beads (20 mg; Macrosorb C, Phase Sep) were measured as described by Cannon et al. (1995b
).
SDS-PAGE and electroblotting.
SDS-PAGE was performed according to the method of Laemmli (1970) through 12·5% (w/v) acrylamide gels. The apparent molecular mass of proteins was estimated using pre-stained protein markers (15·4200 kDa; Gibco-BRL). Sample protein concentrations were determined using a modified Bradford assay (Bio-Rad). Proteins were electroblotted (Towbin et al., 1979
) onto nitrocellulose membranes (Hybond-C; Amersham) for use in blot overlay assays. Transfer conditions were 100 V, 1·5 h, at 4 °C and membranes were stored in plastic wrap at 4 °C until use.
Blot overlay assay.
The binding of C. albicans ATCC 10261 cells to electrophoretically separated parotid saliva proteins, immobilized on nitrocellulose membranes, was determined as previously described (OSullivan et al., 1997 ). Salivary proteins on blot membranes were first visualized by staining them with 0·2% (w/v) Ponceau S in 1% (v/v) acetic acid for 5 min, followed by four washes with 1% (v/v) acetic acid. The membranes were destained with two washes (30 min, 20 °C) of PBS (l-1: 8·5 g NaCl, 0·3 g KH2PO4, 0·6 g Na2HPO4, pH 7·5) and blocked with 5% (w/v) BSA in KCl buffer (2 h, 20 °C). Membranes were incubated with radioactively labelled C. albicans cells [30 ml, 1·1x107 cells (ml KCl buffer)-1] for 18 h at 4 °C with reciprocal shaking (70 min-1), washed four times in PBS containing 0·1% (v/v) Tween 20 to remove non-specifically bound C. albicans cells and air-dried prior to exposure to X-ray film.
Fluid-phase adsorption assay.
Proteins were adsorbed from parotid saliva by microbial cells following a modification of the method described by Newman et al. (1993) . Streptococcal or candidal cells were harvested from cultures by centrifugation, washed with KCl buffer as described above, and suspended in KCl buffer at a concentration of 8x107 streptococcal cells ml-1 or 4·4x107 C. albicans cells ml-1. Bacterial or yeast cell suspensions (0·3 ml) were added to parotid saliva diluted to 0·5 mg protein ml-1 with KCl buffer (0·6 ml) and incubated with end-over-end rotation (9 r.p.m.) at 20 °C for 1 h. Cells were sedimented by centrifugation (2900 g, 4 °C, 10 min) and the supernatant was collected and analysed by SDS-PAGE or retained as pre-adsorbed parotid saliva samples for use in blot overlay and adhesion assays.
Coadhesion assay.
C. albicansstreptococcal co-adhesion assays were performed essentially as described by Holmes et al. (1995a , 1996
). Streptococcal or candidal cells were washed and suspended at a concentration of 4x108 cells ml-1 or 4·4x107 cells ml-1, respectively, in TNMC (1 mM Tris/HCl, pH 8·0, containing 0·15 M NaCl, 1 mM MgCl2 and 1 mM CaCl2). Bacterial or yeast cell suspensions (50 µl) were dispensed into 96-well microtitre plates (Scintistrip, Wallac Oy), centrifuged (800 g, 5 min, 20 °C) to immobilize cells, and the supernatant was discarded. The wells were washed twice [150 µl, 0·05%, v/v, Tween 20 in TNMC buffer (Tween-TNMC)] and incubated with Tween-TNMC (150 µl, 16 h, 4 °C). The liquid was aspirated from the wells and 50150 µl portions of saliva samples (diluted in TNMC buffer) were added and incubated at 20 °C for 1 h. The wells were washed twice (150 µl Tween-TNMC) and then incubated with radiolabelled C. albicans cells [0·1 ml, 2·5x106 cells (ml TNMC buffer)-1, 20 °C, 2 h, with shaking (50 r.p.m.)]. Liquid was aspirated from the wells, which were washed four times (150 µl Tween-TNMC, 10 min) before allowing them to dry (37 °C, 1 h). The plastic well strips were snap-separated and bound radioactivity was counted using a 1450 Microbeta Plus liquid scintillation counter (LKB, Wallac Oy).
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RESULTS |
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Proteins bound by streptococci also support adhesion of C. albicans to experimental pellicle
To determine if the same salivary components that promote binding of C. albicans to streptococci also promote adherence of Candida cells to saliva-treated HA beads, parotid saliva samples were pre-treated with streptococcal cells and then used to coat HA beads. There was a >50% reduction in the numbers of C. albicans cells binding to HA beads coated with saliva that had been pre-incubated with S. gordonii cells, compared with untreated saliva (Fig. 3). A reduction (28%) was also noted in numbers of yeast cells adhering to S. oralis-treated saliva-coated beads, but there was no significant effect on the numbers binding to HA beads coated with saliva that had been pre-incubated with S. mitis, S. mutans or C. albicans (Fig. 3
).
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DISCUSSION |
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Salivary proteins and glycoproteins are present in multiply active conformations within the human oral cavity. In the fluid phase, salivary components may be present as discrete molecules or more usually as macromolecular complexes often involving mucins (Iontcheva et al., 1997 ). A number of these components are selectively adsorbed onto enamel, and the aPRPs and statherin change conformation upon binding to enamel (Moreno et al., 1984
) thus unmasking receptors for adhesion of streptococci and Actinomyces species (Gibbons & Hay, 1988
; Gibbons et al., 1990
, 1991
). This ability of oral micro-organisms to bind immobilized salivary proteins is of considerable ecological significance. Adhesins that have evolved to bind immobilized conformations of salivary components provide a mechanism for attachment of microbial cells to oral surfaces despite the presence of excess soluble forms of the receptors within saliva. The bPRPs appear to comprise another group of salivary proteins that act as receptors for microbial cell adhesion when immobilized. Interestingly, the streptococci, in particular S. gordonii, are able to adsorb these proteins to their surfaces and the bound forms then act as receptors for C. albicans adhesion. Conversely, C. albicans cells are unable to adsorb bPRPs from saliva, although small amounts of other macromolecules are adsorbed by yeast cells (Edgerton et al., 1993
). Thus C. albicans demonstrates a unique adhesive mechanism whereby adhesion may occur to surface-bound forms of bPRPs despite the presence of excess fluid-phase bPRPs in saliva.
Streptococci are early colonizers of salivary pellicles and their ability to bind salivary proteins and glycoproteins is important in plaque development. Of the streptococcal species tested, S. gordonii cells bound salivary proteins to the greatest extent, but the components of saliva bound by the streptococcal species varied quite considerably. The changes to the salivary protein profiles observed following incubation with streptococcal cells were highly reproducible, and evidence suggested that the alterations resulted from adsorption of the proteins to the bacterial cell surface rather than for other reasons such as proteolysis. Even in the presence of a cocktail of protease inhibitors, bPRP bands A, C and D were adsorbed by S. gordonii within 15 min determined by the overlay assay (data not shown). Previously, S. gordonii cells have been reported to bind to PRPs IB-1 to IB-9 and Ps-1 in blot overlay assays (Newman et al., 1996 ). The adsorption of bPRPs by S. gordonii occurs via a high-affinity mechanism since maximal adsorption of bPRPs from the fluid phase occurred under conditions that did not result in the complete removal of
-amylase, which is a high-affinity binding reaction (Scannapieco et al., 1989
). Pool 4 promotes high binding of C. albicans to immobilized S. gordonii cells and it is possible that all four bPRPs are recognized, when presented on the surface of the bacterial cells, with a concomitant increase in binding activity. As a result of adsorption of bPRPs by streptococci, the PRP-depleted saliva was much less efficient at supporting C. albicans adhesion to HA beads, confirming that similar forms of the adhesion receptors are presented by enamel and by streptococci.
S. gordonii is found at multiple sites within the human oral cavity and is associated with numerous intra- and inter-generic bacterial co-aggregations which are postulated to be involved in the formation of oral biofilms (Kolenbrander & London, 1993 ; Holmes et al., 1996
). Therefore, adhesion of C. albicans cells to salivary components adsorbed to S. gordonii cells would potentially increase the number of available sites for C. albicans adhesion and colonization. In support of this suggestion, it has been observed that C. albicans cells are more usually found associated with plaque-coated areas of enamel (Arendorf & Walker, 1980
). It could be envisaged that presentation of bPRPs on the surface of streptococci might also tend to co-aggregate C. albicans and promote clearance from the oral cavity. However, streptococcal cells also bind to aPRP 1-coated enamel (Gibbons et al., 1991
). Thus direct co-aggregation of C. albicans with streptococci, or co-aggregation via bPRP, followed by binding of streptococci to an oral surface could also provide a means by which C. albicans colonization is promoted.
Currently, the molecular nature of the receptor that binds bPRPs to the streptococcal cell surface is not known. However, two protein families on the streptococcal cell surface are known to bind to other salivary proteins. The antigen I/II proteins (Jenkinson & Demuth, 1997 ) bind salivary agglutinin glycoprotein while AbpA (Rogers et al., 1998
) binds
-amylase. The antigen I/II proteins SspA and SspB are also involved in the direct binding of S. gordonii to C. albicans cells (Holmes et al., 1996
). We have observed that mutants deficient in the production of SspA and SspB are reduced, by approximately 20%, in their ability to adsorb bPRPs (unpublished observations). However, since adsorption of bPRPs by wild-type cells appears to be a rapid and high-affinity process, simply reduced adsorption by the mutants implies that the SspA and SspB proteins are not the major bPRP adhesins. Therefore, it seems likely that the binding of bPRPs to the streptococcal cell surface may occur through a multi-modal mechanism.
In summary, C. albicans cells do not appear to bind salivary bPRPs in the fluid phase. However, adsorption of these proteins to a surface such as enamel, Streptococcus, or to nitrocellulose in vitro exposes receptors that are then recognized by C. albicans cells. This ability to adhere to only surface-bound peptides would enable C. albicans cells to adhere to multiple oral cavity surfaces in the presence of fluid-phase saliva and thus enhance its colonization potential.
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ACKNOWLEDGEMENTS |
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Received 2 August 1999;
revised 28 September 1999;
accepted 14 October 1999.