Interaction of the salivary low-molecular-weight mucin ... - Springer Link

Antonie van Leeuwenhoek 70: 79-87. 1996. 9 1996 KluwerAcademic Publishers. Printedin the Netherlands.

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Interaction of the salivary low-molecular-weight mucin (MG2) with ActinobaciUus actinomycetemcomitans J. Groenink*, A.J.M. Ligtenberg, E.C.I. Veerman, J.G.M. Bolscher & A.V. N i e u w A m e r o n g e n Department of Oral Biochemistry, Academic Centrefor Dentistry Amsterdam (A CTA), the Netherlands (*author for correspondence) email: J. Groenink.obc.acta @reed.vu.nl Received9 October1995;Acceptedin finalform27 February1996

Key words: Actinobacillus actinomycetemcomitans, adherence, mucins, periodontitis, saliva, sialic acid

Abstract Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.

Abbreviations: Secretory I g A - S-IgA; high-molecular-weight mucin - MG 1; low-molecular-weight mucin - MG2; extra parotid-glycoprotein - EP-GP; proline-rich proteins - PRPs; Sambucus nigra agglutinin - SNA; Maackia amurensis agglutinin - MAA; peanut agglutinin - PNA; Ulex europaeus agglutinin - UEA

Introduction Periodontitis is characterized by the destruction of the supporting tissues of the teeth and resorption of the alveolar bone structure. One of the microorganisms associated with the occurrence of adult and juvenile periodontitis is Actinobacillus actinomycetemcomitans (Slots & Listgarten 1988; Van Winkelhoff & de Graaff 1991; Zambon 1985). In this respect, the adherence of this bacterium to oral surfaces is probably an initial and essential step in the pathogenesis of periodontitis. Adhesion of bacteria to oral surfaces is often mediated by specific interactions between carbohydrate residues of receptor glycoproteins on host tissues and lectinlike proteins, so-called adhesins, on the bacterial cell surfaces (Ofek & Perry 1985).

Saliva may influence the formation of microbial complexes onto the oral surfaces in a dualistic way: by interacting with the bacterial surface adhesins salivary components block the interaction with receptors on the host tissues (Williams & Gibbons 1972). In addition, they may mediate the aggregation and clearance of oral bacteria (Ligtenberg et al. 1992; Ligtenberg et al. 1993; Liljemark et al. 1979). On the other hand, salivary proteins present in the protein pellicles covering the tooth and mucosal tissues, can function as additional receptor sites for bacterial adhesins, thereby enhancing bacterial colonization on the oral surfaces (Kishimoto et al. 1989). In this context, binding of various salivary components to microorganisms has been demonstrated for a number of salivary proteins, e.g. high-molecularweight mucins (MG1) (Veerman et al. 1995), low-

80 molecular-weight mucins (MG2) (Hoffman & Haidaris 1993; Levine et al. 1987; Ligtenberg et al. 1992; Stinson et al. 1982; Tabak et al. 1982), proline-rich glycoproteins (Gillece-Castro et al. 1991; Shibata et al. 1980), c~-amylase (Scannapieco et al. 1989), secretoryIgA (S-IgA) (Brandtzaeg et al. 1968; Bratthall & Carlen 1978; Liljemark et al. 1979; Williams & Gibbons 1972), parotid agglutinin (Rundegren 1986) and the extra-parotid glycoprotein (EP-GP) (Schenkels et al. 1993). For A. actinomycetemcomitans binding to specific immunoglobulins has been demonstrated in human saliva and serum (Engstrrm et al. 1993; Nakagawa et al. 1994; Sandholm et al. 1987; Saxrn et al. 1990). Furthermore, it has been demonstrated that a salivary pellicle inhibits binding of A. actinomycetemcomitans to hydroxyapatite (Kagermeier & London 1985). More recently, it has been indicated that binding of human saliva to A. actinomycetemcomitans inhibits the adhesion of the bacterium to a human oral cell line (Mintz & Fives-Taylor 1994). The salivary components involved in these inhibitory effects have not been identified yet. In the present study, we have investigated the interaction of A. actinomycetemcomitans with salivary components, both in the liquid phase and the solid phase, i.e. immobilized onto nitrocellulose membranes. The results indicate that particularly the low-molecular-weight mucin MG2, parotid agglutinin, and a 300 kDa submandibular/sublingual glycoprotein bind to the bacterium.

Materials and methods

Materials Neuraminidase (type VIII) from Clostridium perfringens, 5-bromo-4-chloro-3-indolyl-phosphate, nitro blue tetrazolium, and the lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), peanut agglutinin (PNA), and Ulex europaeus agglutinin (UEA) were obtained from Boehringer (Mannheim, Germany). Sodium borohydride, sodiummeta-periodate and Schiff's reagent were from Merck (Darmstadt, Germany). Bovine serum albumin (BSA), Coomassie Brilliant Blue R-250 and Tween 20 were from Sigma Chemical Co. (St. Louis, Mo.). 4-chloro1-naphthol was from Pierce Chemical Co. (Rockford, I11.). Sodium dodecyl sulfate-polyacrylamidegel electrophoresis high-range molecular protein standards were from BioRad (Hercules, Ca.). Monoclonal antibodies 150AA1.1,141AA1, and 261AA2.2 specific for

serotypes a, b, and all serotypes of A. actinomycetemcomitans strains, respectively, were kindly provided by Dr R. Gmiir (Ziirich, Switzerland). Rabbit antibodies to the a-chain of human IgA, to the secretory component of S-IgA, to a-amylase, alkaline phosphataseconjugated and peroxidase-conjugated goat anti-rabbit immunoglobulins and rabbit anti-mouse immunoglobulins were obtained from Dako (Glostrup, Denmark). Mouse monoclonal antibodies 7E5 to EP-GP (Rathman et al. 1990; Schenkels et al. 1993), E9 to a sialic acid containing epitope on glycoproteins (Rathman et al. 1990; Veerman et al. 1991), F2 to MG1 (Rathman et al. 1990), and antibodies to cystatins S and C (Henskens et al. 1994) have been described elsewhere. A polyclonal antibody raised to a synthetic peptide of the C-terminal region of MG2 was used (J.G.M. Bolscher et al., unpublished results). Anti-proline-rich proteins (PRPs) was a kind gift of Dr M. J. Levine (Buffalo, NY). Nitrocellulose was from Schleicher and SchiJll (Dassel, Germany). The electrophoresis equip~ ment and materials (PhastSystem) were obtained from Pharmacia (Uppsala, Sweden).

Culturing and harvesting of bacteria Three strains ofA. actinomycetemcomitans were used: HG 1175 (serotype a), Y4 (serotype b), both obtained from Dr A. J. van Winkelhoff (Amsterdam, The Netherlands), and NY 644 (serotype b), obtained from Dr J. S. van der Hoeven (Nijmegen, The Netherlands). All strains were isolated from periodontal lesions of patients with periodontitis. Strains were cultured anaerobically to maintain the fimbriae bearing surface of the bacterium. The smooth laboratory strain Y4 was cultured aerobically. The bacteria were stored in Protect bacterial preservers (Technical Service Consultants Ltd., Bury, Lancashire, UK) at - 80 ~ until use. For each experiment, cells were cultured on sheep or horse blood agar plates and grown for 48 hr at 37 ~ under anaerobic or aerobic conditions with 5 % CO2. Rough colonies of the clinical isolates HG 1175 and NY 644 were picked up and inoculated onto fresh blood agar plates. Bacteria were harvested after 48 hr at 37 ~ by wiping off the agar plates with sterile cotton swabs and were transferred to phosphate buffered saline (PBS, containing 0.15 M sodium chloride and 0.01 M potassium phosphate buffer pH 7.4). Bacteria were then collected by centrifugation at 3,000 g for 10 min and washed in 10 ml PBS. After centrifugation, the pellet was resuspended in PBS by gently stirring using a

81 vortex mixer. The concentration of the suspension was adjusted to an optical density at 700 nm (OD70o) of 1.0.

Collection of human whole and glandular salivas Human whole saliva (HWS) was collected by expectoration without stimulation in ice-cooled vessels. Parotid (Par) saliva was collected by use of a Lashley cup under stimulation with a solution of 2 % citric acid or by chewing paraffin. Saliva samples from the sublingual (SL) and the submandibular (SM) glands were collected separately without stimulation by a custommade, individually adapted saliva segregator (Schneyer 1955). All saliva samples were stirred on a vortex mixer for 1 min. By this treatment, viscosity of saliva was diminished (Veerman et al. 1989) enabling a subsequent clarification by centrifugation (1,200 g, 10 min at 4 ~ The resulting cleared supernatant was stored in aliquots at - 20 ~ until used to study binding with

A. actinomycetemcomitans. In the liquid-phase binding assay and the solidphase binding assay whole and glandular saliva samples of six healthy subjects were examined (two individuals had blood group A, two blood group B, and two were non-secretors of their blood group A).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed with PhastSystem (Pharmacia, Uppsala, Sweden) SDS-PAGE 4 to 15 % gels. Saliva samples (approximally 1 mg/ml protein) were dissolved in reducing sample buffer (0.05 M Tris buffer, pH 6.8, containing 2 % SDS and 10 mM DTT) according to the standard procedure as described in the PhastSystem manual. Polyacrylamide gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 (CBB) in 30 % (v/v) methanol and 10 % acetic acid and destained in 10 % (v/v) acetic acid. Glycoproteins were stained with periodic acid Schiffs (PAS) reagent as described by Van Seuningen and Davril (1992).

Liquid-phase binding assay To examine which salivary components interact with

A. actinomycetemcomitans, 200 #1 of a suspension of the bacterium (ODTo0=l.0) in PBS was centrifuged. The obtained pellet was resuspended in 100 #1 saliva and incubated for 1 hr at 37 ~ Thereafter, bacteria were collected by centrifugation (3,000 g for 10

min) and washed in PBS. Following centrifugation (10,000 g for 2 min) cell-bound salivary components were solubilized by incubating the obtained pellet in 0.5 % SDS in PBS for 30 min at 4 ~ The bacterial extracts were examined by SDS-PAGE on 4 to 15 % polyacrylamide gels. Control extracts were obtained from bacteria that had not been incubated with saliva. After SDS-PAGE, proteins were blotted onto sheets of nitrocellulose by means of diffusion blotting (2 hr), according to a method described by Braun and Abraham (1989). Nitrocellulose membranes were probed with various mono- and polyclonal antibodies to several salivary proteins, e.g. MG1, MG2, IgA, amylase, and cystatins. After the blots were washed, bound antibody was detected by alkaline phosphatase conjugated to either goat anti-rabbit immunoglobulins or rabbit anti-mouse immunoglobulins with 5-bromo-4-chloro3-indolyl-phosphate and nitro blue tetrazolium used as substrates.

Solid-phase binding assay Attachment of bacteria to salivary proteins immobilized on nitrocellulose was conducted by an overlay blotting technique according to Prakobphol et al. (1987). In short, saliva samples (1 mg/ml protein concentration) were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were blocked with a solution of 3 % BSA and 0 . 1 % Tween 20 in PBS (PBS-T-BSA) for 1 hr at ambient temperature. A. actinomycetemcomitans, resuspended in PBST-BSA to ODT0o of 0.5, was incubated with the blots for 18 hr at 4 ~ Blots were then washed twice with PBS and once with PBS-T-BSA, whereafter bound bacteria were probed with murine monoclonal antibody directed to different serotypes of A. actinomycetemcomitans (Gmiir & Guggenheim 1990). Antibodies 150AAI.1, 141AA1, and 261AA2.2 were used to detect A. actinomycetemcomitans strains HG 1175, NY 644, and Y4, respectively (Gmiir & Guggenheim 1990). After repeated washing with PBS containing 0.1% Tween (PBS-T) bound antibody was detected by horse-radish peroxidase conjugated rabbit-anti-mouse immunoglobulin with 4-chloro-l-naphthol and H202 used as substrates. Blots, exposed to the secondary antibody only, showed no staining. To oxidize carbohydrate residues on glycoproteins blots were incubated with sodium-meta-periodate (0.02 M) as described by Woodward et al. (1984). Removal of sialic acid was achieved either after electrophoretic separation, by incubating blots 45 min at 80

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Table 1. Effect of chemical and neuraminidase treatment of SL saliva on the carbohydrate structures of MG2, determined on western blot by lectin binding. Lectins

Sambucus nigraagglutinin (SNA) Maackiaamurensis agglutinin (MAA) Peanut agglutinin (PNA) Ulex europaeus agglutinin (UEA)

Sugar specificity a(2-6) N-acetylneuraminicacid ct(2-3) N-acetylneuraminicacid D(+)galactose a-L(-)fncose

Lectin binding toMG2after treatment* Control Neuraminidase Mild acid

Periodate

+ + + +

0 0 -

++ +

0 ++ -

*Scoring of lectin binding: + = detected; 0 = not detectable; ++ = increased; - = decreased.

~ under mild acidic conditions (0.05 M HC1, pH 2.0), or before electrophoresis, by incubating saliva with 0.5 U/ml neuraminidase (type VIII) from Clostridium perfringens in 0.1 M acetate buffer, pH 5.5, at 37 ~ for 16 hr. The effects o f the various treatments on the carbohydrate moiety of M G 2 was determined by the lectins SNA, M A A , PNA, and U E A specific for the carbohydrates a ( 2 - 6 ) N-acetylneuraminic acid, linebreak a ( 2 - 3 ) N-acetylneuraminic acid, D(+)galactose, and a - L ( - ) fucose, respectively (Table 1).

Results

Identification of salivary components interacting with A. actinomycetemcomitans Salivary components interacting with the strain HG 1175 of Actinobacillus actinomycetemcomitans were identified in a liquid-phase binding assay. In these experiments whole salivas and salivas from the parotid, sublingual, and submandibular glands o f six subjects were incubated with A. actinomycetemcomitans and after extraction adsorbed proteins were analysed by SDS-PAGE and immunoblotting with antibodies to several salivary proteins (Table 2). The salivary proteins a - a m y l a s e , cystatins S and C, proline-rich proteins (PRPs), and EP-GP did not bind to A. actinomycetemcomitans H G 1175. Only a small fraction of the total salivary I g A bound to A. actinomycetemcomitans. Repeated incubation o f the supernatant fraction with fresh bacteria did not result in further depletion o f S-IgA. Saliva from one individual showed no binding o f S - I g A to A. actinomycetemcomitans. Collectively, these results suggested that only a specific subset of salivary I g A binds to antigens present on A. actinomycetemcomitans, in line with previous reports (Engstr6m et al. 1993; Nakagawa et al. 1994; Sandholm et al. 1987; Sax6n et al. 1990).

Table 2. Binding of salivary proteins and glycoproteins from CHWS and glandular salivas to Actinobacillus actinomycetemcomitans HG 1175 as determined in the liquid-phase binding assay. Saliva component

Salivary component found in 1 Bacterial PAR SL SM extract2

a-Amylase Cystatin C Cystatin S EP--GP S-IgA MG1 MG2 PRP Parotid agglutinin 300 kDa glycoprotein

+ + -

+ +

+ + + + -

+ + + + +

+ + + + + + + + +

1The presence of several salivarycomponentsis characteristic for the glandular saliva of which they are derived from. 2Extract is the bacterium-bound saliva fraction as obtained from the liquid-phase binding assay. In this assay different salivas of the parotid (PAR), sublingual (SL), and submandibular (SM) glands (n = 6) were used. The presence of a salivary componentis indicated by + and the absence by -.

When in the liquid-phase assay nitrocellulose filters were probed with anti-mucin m A b E9, recognizing a sialic acid-containing epitope on the salivary glycoproteins, the low-molecular-weight mucin M G 2 and a 300 kDa sialic acid-containing glycoprotein, present in both sublingual and submandibular salivas (Table 2), bound to H G 1175 (Figure 1, lanes 3 & 5) and could be depleted from saliva by repeated incubation with fresh bacteria (Figure 1, lane 4). Probing with a polyclonal antiserum elicited against the C-terminal peptide region o f MG2, confirmed the bacterial binding and depletion of M G 2 (Table 2). In parotid salivas the binding o f the 300 kDa agglutinin to H G 1175 was observed, either detected by m A b E9 or anti-Lewis (Y) antibody (Takano et al. 1992) (Table 2). Figure 1 fur-

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A. actinomycetemcomitans adherence to electrophoretically separated salivary proteins

Figure 1. Depletionof salivarymucins from sublingual (SL) saliva by A. actinomycetemcomitansshownon western blot. A nitrocellulose replica of a 4-15 % SDS-polyacrylamidegel shows SL saliva (lane 1), SL supernatant after incubation with HG 1175 (lane 2), bacterial extract after incubation with SL saliva (lane 3), secondary supernatant afterreincubationwith freshbacteria(lane4), secondary extract afterincubation of bacteriawith SL supernatant (lane 5). The salivaryproteinsMGI, MG2,and a 300 kDa glycoproteinare detected by mAb E9, directedto a sialic acid containingepitopeon salivary glycoproteins(37). Lane6 showsthe SDS extractof the bacterianot incubated with saliva. The positionof the molecularmassmarkersis given on the left. The position of the high-molecular-weightmucin MG1, a 300 kDa glycoprotein,and the low-molecular-weightmucin MG2 is indicated on the fight.

ther shows that the high-molecular-weight mucin MG1 could notbe detected in the bacterial extracts (lanes 3 & 5), although some reduction was noted in supernatant (lanes 2 & 4). Probing nitrocellulose replicas with antiMG1 mAb F2 confirmed that MG1 was not present in the bacterial extracts (Table 2). With this antibody no decrease in the M G I signal was observed in the supernatant fraction. The smooth laboratory strain Y4, tested in the liquid-phase binding assay, bound also MG2, parotid agglutinin, and the 300 kDa sublingual and submandibular glycoprotein (not shown). Extracts of bacteria, which were not incubated with saliva, showed little or no non-specific binding of antibodies used (Figure 1, lane 6).

The interaction between A. actinomycetemcomitans and salivary proteins was investigated further in the solid-phase binding assay (Prakobphol et al. 1987). In this assay, sublingual saliva, after electrophoresis, was blotted onto nitrocellulose, which was subsequently incubated with the strains HG 1175, NY 644, or Y4. Figure 2A, lane 1 shows the electrophoretic pattern after double staining of the (glyco)proteins with CBB and PAS. The PAS-positive band at 180 kDa position was identified as MG2, by immunoblotting with the anti-MG2 polyclonal antiserum (Figure 2A, lane 3). After incubation of nitrocellulose replicas with suspensions ofA. actinomycetemcomitans, only adherence to MG2 was observed (Figure 2A, lane 4, HG 1175; lane 5, Y4). Essentially all strains tested bound to MG2 and the same results were obtained with all sublingual salivas (6 subjects) tested. No adherence at all was found to parotid proteins in the solid-phase binding assay (not shown). The molecular structures underlying the attachment A. actinomycetemcomitans to MG2 have been examined in more detail in the solid-phase binding assay. First, the nitrocellulose membranes of the solidphase binding assay were incubated with sodium-metaperiodate to oxidize carbohydrate residues or, under mildly acidic conditions to hydrolyse terminal sial: ic acid residues. Following these treatments, binding of MG2 by the strain HG 1175 was completely abolished (Table 3), suggesting that carbohydrates and most probably sialic acid residues, are involved in the binding of MG2 to HG 1175. To investigate this further, sublingual salivas were incubated with neuraminidase from Clostridium perfringens (Figure 2B). It appeared that asialo-MG2 migrated more slowly into the SDS-polyacrylamide gel (compare Figure 2A, lane 1 with 2B, lane 1). Western blot detection with the anti-MG2 polyclonal antiserum, directed to the C-terminal peptide part of MG2, confirmed this shift (Figure 2B, lane 3). The effectivity of the neuraminidase treatments was verified by immunoblotting with a panel of lectins (Table 1). Only a slight residual activity of a(2-3) N-acetylneuraminic acid and a(2--6) N-acetylneuraminic acid residues on neuraminidase, treated MG2 was observed (compare Figure 3A and B, lanes 3 & 4). Whilst as a result of desialylation the binding of lectin PNA to penultimate galactose residues on MG2 was increased (Figure 3, lane 2).

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Figure 2. Binding of A. actinomycetemcomitans HG 1175 to electrophoretically separated MG2 before and after neuraminidase treatment. Sublingual salivary samples were electrophoretically separated by SDS-PAGE (4--15 %), transferred to nitrocellulose, and overlaid with bacteria. After washing, bacteria were probed with polyclonal antibodies to A. actinomycetemcomitans. Position of molecular markers is indicated on the left. Both lanes 1 show the double-stained Coomassie Brilliant Blue- and PAS-stained bands of sublingual saliva before (A) and after removal of sialic acid residues by neuraminidase of Clostridiumperfringens (B). Corresponding, lanes 2 and 3 show the immunodetection of, respectively, MG1 by mAb F2 and MG2 by the polyclonal antiserum directed to the C-terminal peptide part of MG2. The detection of A. actinomycetemcomitans strains HG 1175 and Y4, adhered to the nitrocellulose replicas, is presented in lanes 4 and 5, respectively. Table 3. Adherence ofA. actinomycetemcomitans to MG2, as determined in the solid-phase binding assay, before and after chemical or neuraminidase treatment of sublingual saliva. Strain 1

Culture condition

Phenotype

MG2 binding2

MG2 binding after variant treatments of sublingual saliva2 Neuraminidase Mild acid Periodate

HG 1 1 7 5 NY 644 FDC Y4

Anaerobe Anaerobe Aerobe

Rough Rough Smooth

+ + +

+

0 ND +

0 ND +

tA. actinomycetemcomitans strain HG 1175 of the serotype a is detected by monodonal antibody 150AAI.1, while strains NY 644 and FDC Y4 of serotype b are detected by 141AAI and 261AA2.2, respectively. 2The bacterial adherence to MG2 is indicated by + to illustrate binding, and - or 0 to illustrate decrease or abolishment of binding, respectively. ND stands for not determined.

In the solid-phase binding assay, the attachment o f the strains H G 1175 and N Y 644 to a s i a l o - M G 2 was a l m o s t c o m p l e t e l y abolished (Figure 2B, lane 4). In contrast, the strain Y 4 still adhered to a s i a l o - M G 2 (Figure 2B, lane 5). T h e binding o f Y 4 was even unaffected after p e r i o d a t e treatment o f M G 2 (Table 3).

In subsequent studies, A. actinomycetemcomitans isolate H G 1175, g r o w n under anaerobic or aerobic conditions, was tested in the solid-phase b i n d i n g assay. Collectively, the different growth conditions and variant cell m o r p h o l o g y o f the bacteria ( H G 1175, serotype a, both r o u g h and s m o o t h colonies; N Y 644, s e r o t y p e

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Figure3. Analysisof the carbohydratestructureson MG2 by lectin detection.Nitrocelluloseblotsof SDS-polyacrylamidegels(4-15 %) containing controlsublingual saliva (A) and neuraminidase-treated SL saliva(B) are immunostainedwith an anti-MG2polyclonalantiserum (lane 1) and the lectins PNA (lane 2), SNA (lane 3), and MAA (lane 4). Neuraminidasetreatment of MG2 led to the exposure of galactose residues and diminishing of NeuAca(2~) and NeuAca(2- 3) residues.

b, rough; Y4, serotype b, smooth) had no influence on the adherence to the low-molecular-weight mucin.

Discussion

In the present study, we have identified the salivary components binding to A. actinomycetemcomitans. Three strains HG 1175, NY 644, and Y4, all isolated from lesions of patients with periodontitis, were used in our study. In solution, A. actinomycetemcomitans adhered to S-IgA, the low-molecular-weight mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein (Table 2). The identity of the 300 kDa glycoprotein detected in sublingual and submandibular saliva remains uncertain, but it is likely to be identical to a glycoprotein described by Kishimoto et al. (1989) and Ebisu et al. (1988). No binding of the salivary proteins MG1, PRPs, amylase, EP-GP, and the cystatins S and C to the bacteria was observed in the liquid-phase assay. In addition, in the overlay blotting assay all three A. actinomycetemcomitans strains tested adhered to only MG2. This is remarkable, because findings of Biesbrock et al. (1991) describe the dependence of MG2 on complexing with secretory IgA to interact with microorganisms. In the present study, MG2 could

apparently bind to A. actinomycetemcomitans by itself. Removal of sialic acid residues on MG2 resulted in a virtually complete abolition of binding to strains HG 1175 and NY 644, indicating that sialic acid-containing oligosaccharides on MG2 function as ligands for the A. actinomycetemcomitans adhesins. Although sialic acid residues are also present on MG1 this protein did not bind to A. actinomycetemcomitans. Analogous results have been obtained in previous studies for the interaction between S. gordonii and salivary mucins (Levine et al. 1987; Ligtenberg et al. 1991). In these studies it was demonstrated that S. gordonii bound to MG2 and not to MG 1, and furthermore that the binding was mediated by sialic acid-containing oligosaccharides of MG2. The difference in affinity towards MG1 and MG2 is not known, but may be clearified when the precise carbohydrate structures of the ligands on MG2 is identified. This is the subject of further studies. Strikingly, the MG2 binding of the strain Y4 was unaffected even after periodate treatment of MG2. The different response exhibited by Y4 and the two other clinical isolates to the enzyme and chemical treatments of MG2 was unexpected. However, distinctive mechanisms of adhesion for strains of A. actinomycetemcomitans, have already been described by Kagermeier and London (1985). The apparent differences between the receptors for MG2 are not reflected in the presence or absence of fimbriae, because suspensions from rough and smooth colony forms of the strain HG 1175 bind MG2 equally well (unpublished results). Mintz and Fives-Taylor (1994) have demonstrated that, in the presence of saliva, adherence of A. actinomycetemcomitans to human oral epithelial cells is inhibited. The results of the present study suggest that in particular MG2, parotid agglutinin, and a 300 kDa glycoprotein, are probably involved in this process. All three glycoproteins expose sialic acid residues, of which the involvement in binding to the bacterium is demonstrated for MG2. Thus, it may be plausible that sialic acid-containing glycoconjugates on epithelial cells may function as receptors for A.

actinomycetemcomitans. In addition, it seems unlikely that MG2 represents a binding site for A. actinomycetemcomitans in the oral cavity as part of the dental pellicle. MG2 has been identified only as component of a young (20 minutes) in vivo enamel pellicle, but is no longer detectable in a 2-h in vivo enamel pellicle (A1-Hashimi & Levine, 1989; Levine et al. 1985). According to our study, components of the acquired enamel pellicle as described in literature, e.g. MG1, PRPs, c~-amylase, and cys-

86 tatins (AI-Hashimi & Levine 1989), did not bind to

A. actinomycetemcomitans. Thus, these components can function as an anti-adhesive barrier for A. actinomycetemcomitans, supporting findings o f Kagermeier and London (1985), that a salivary protein layer on hydroxyapatite inhibits the attachment o f A. actino-

mycetemcomitans. On the other hand, M G 2 has been detected bound to the cementum, the tissue layer which attaches the tooth to the alveolar bone (Fisher et al. 1987). The presence o f M G 2 in this site may favor the colonization o f A. actinomycetemcomitans to root cementum exposed as a result o f periodontitis. Studies under more physiological environmental conditions, like adherence to epithelial cells and teeth, must be performed in the future to determine the precise role o f saliva in the colonization process o f the bacterium. M G 2 binding to H G 1175 and NY 644 was dependent on the presence o f sialic acid residues on the mucin. In this context, it has to be noted that during periodontal inflammation and poor oral hygiene, probably as a result o f increased bacterial neuraminidase activity, the amount of free sialic acid has been increased in the oral cavity (Gibbons et al. 1990; Nieuw A m e r o n g e n et al. 1992; Pertsch & Glickman 1967). It is conceivable that under these conditions the sialic acid content of M G 2 will be diminished, and consequently its bacteria-binding properties will be impaired. It may be speculated that as a result of these conditions the oral colonization o f various bacteria, among these A. actinomycetemcomitans, can be facilitated. Summarising, in the present study binding of salivary components to A. actinomycetemcomitans was observed for the glycoproteins MG2, parotid agglutinin, and a sublingual/submandibular 300 kDa glycoprotein. The specific binding of M G 2 to two clinical isolates is orientated through sialic acid residues. MG2 binding by strain Y4 is probably determined by a peptide domain on MG2. This work supports the concept that mucins play an important role in the protection of oral surfaces from the daily threat of bacteria (Levine et al. 1985; Tabak et al. 1982).

Acknowledgments We thank Miss J. S. van der Kwaak for technical assistance. We acknowledge especially Dr R. Gmtir for providing the monoclonal antibodies to A. actinomycetem-

comitans.

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