Scand.J. DenL Res.. 1977: 85: 387-39J (K.i:.T words: denial plaque, hydroxyapatite. Streptococcus mutansi

Adsorption of Streptococcus mutans lipoteichoic acid to hydroxyapatite J,E,CIARD1,G. ROLLA, W, H. BOWEN ANDJ. A. REILLY Etiology Section, Caries Prevention and Research Branch, National Canes Program N.I.D.R,, N.l.H,, Bethesda, Maryland, U.S.A.

ABSTRACT — Lipoteichoic acid extracted from cells of S, mutans strain BHT exhibited a high affinity for hydroxyapat:ite. Phosphate ions, fluoride ions and to a lesser extent human saliva inhibited or reversed this adsorption. Extracellular lipoteichoic acid preparations obtained from the supernatant of cultures of the same bacteria exhibited similar properties. It is suggested that lipoteichoic acids could play a significant role in the colonization of teeth by Gram-positive bacteria and thereby contribute to the formation and pathogenicity of dental plaque, (Accepted for publication 18 December 1976)

Acidic molecules such as casein, polyglutaraate, pol^'phosphates and organic phosphates have been shown to possess high affinity for hydroxyapatite and dental

coccus mutans a n d Streptococcus sanguis produce significant amounts of extracellular teichoic acid (MELVAER et al. 1974,

enamel

(BERNARDI

1968,

MARKHAM

PRUITT,

JAMISON

1970,

SHOCKMA.N' 1975, CHIU, EMDUR &: PLATT

1 9 7 0 , N O R D B O SC

1974) and it seems conceivable that the binding of 5. mutans to teeth in the presence of dietary sucrose may, in part, be mediated by complexes of teichoic acid and insoluble polysaccharides in the bacterial coat (R6LLA 1976). These insoluble polysaccharides have been shown to contain bound phosphate

& KAWASAKI &

ROLLA & MATHIESE.N

CALDWELL

ROLLA 1972). The bacteria which initially colonize the tooth surfaces are Grampositive cocci (RiTZ 1967, CARLSSON 1968, KiLlAN & ROLLA 1976). These bacteria apparently possess strongly negatively charged phosphate polymers (lipoteichoic acid) exposed on their surfaces (WlCKEN & KNOX 1975). It has been postulated that lipoteichoic acid possesses physicochemical groups responsible for the rapid adhesion of Gram-positive bacteria to teeth in vivo (MELVAER, HELGELAND & ROLLA

1974, MARKHAM,

KNOX, WICKEN SC HEWETT 1973). Strepto-

(KELSTRUP

et

&

al.

1975,

JOSEPH

FUNDER-NIELSEN

&

1972,

MELVAER et al. 1974), and to be strongly negatively charged and capable of binding calcium

(KELSTKUP

& FUNDER-NIELSEN

1972). The surface of the coat of sucrosegrown S, mutans will thus presunaably

388

CIARDI,, ROLLA, BOWEN AND REILLY

possess a relatively large density of phosphate groups, which may explain its extraordinary affinity for tooth surfaces. The present study was carried out to determine the degree of adsorption of radioactive lipoteichoic acid to hydroxyapatite and to investigate the influence of saliva, Huoride and phosphate ions on this phenomenon.

serum (titer: 1,280) to the L casei lipoteichoic acid. Following hydrolysis of the radioactive preparations in 2 N HCl at 100°C for 3 h they were found to contain phosphate, as measured by the method of FISKE SC SUBBAROW (1925),

and glycerol, by use of the Glycerol Stat-Pacl (Calbiochem Inc.). Radioactive lipoteichoic acid polymers present in culture supernatants (approximately 2,500 c.p.m./jig of glycerol derived from poijoners) were concentrated approximately 50-fold on PM 10 ultrafiltration membranes (Amicon Corp.). Diafiltration of Material and methods the retentates was carried out twice with 50 vol buffer (pH 6.5) containing 10 mM imidazoleBACTERIA HCI and 0.04% NaN, and the final retentate .S. mmtans strain BHT, serotype b (BRATTHALL clarified by centrifugation at 30,000 xg. The 1970) was grown overnight in an atmosphere supernatants also contained glucosyltransof 9596 nitrogen and 5% carbon dioxide at ferase and fructosyltransferase activities, as 37°C in a medium (pH 7.2) prei'iously measured by specific incorporation of r e described by DoNKERSLOOT, ROBRISH & labeled glucosyl- or fructosyl-moieties of sucrose into polysaccharides, KRICHEVSKY (1972) but modified to contain Hydroxyapatite (Bio-gel HTP) was obtained O.O59S NajCOj, 0.2% glucose as carbohydrate Bio-Rad Laboratories, Richmond, source, and 0.5 mCi of glycerol-2-'H (New from England Nuclear Corp., 0.46 mg/mCi) per California, The Ca/P rado was between 1,4—1.5 liter. Cells were removed from the culture by and the surface area 50—75mVg. Results from centrifugation at 16,000 xg and washed three preliminary' experiments, had shown that times with distilled water. Sufficient sodium lipoteichoic acid was adsorbed rapidly by azide was added to the final- 16,,000xg super- hydroxyapatite: the adsorption isotherms were therefore based on 15-min interaction. Varying natant to a concentration of 0;04% and stored concentrations of labeled lipoteichoic acid preat4°C. paration (254 c.p.m./^g dry weight) dissolved in 2 ml of 1 mM phosphate buffer (pH 6.5) LIPOTEICHOIC ACID PREPARATION were stirred with 2 nag hydroxyapatite and Lipoteichoic acid was extracted from cells by centrifuged for 5 min at maximum speed in the vigorously mixing equal volumes of washed International Clinical Centrifuge (International cells suspended in distilled water (in 2% of Equipment Co.), The uptake of lipoteichoic original growth volume) and 90% aqueous acid per mg hydroxyapatite was calculated phenol at 4°C for 2 h. After centrifugation at from the loss of radioactivity in the super20,000 xg the aqueous layer was removed and natant. Radioactivity from 1 ml of the superthe phenol layer re-extracted with 1 vol natant solution was counted with 10 m! distilled water. The aqueous layers were Instagel (Packard Instrument Co.) in a Packard combined and dialyzed three times against Tricarb scintillation counter. Saliva-treated 100 vol distilled water. The resulting pre- hydroxyapatite was prepared as described preparations contained nondialyzable radioactive viously (ROLLA 1971). material (254.167 c.p.m./mg dry weight) and gave a precipitin line in Ouchterlony analysis against antiserum raised against LactobacUltts Results casei lipoteichoic acid. The antiserum, generously provided by Dr. K, W KNOX, is specific for the polyglyceroi phosphate Lipoteichoic acid extracted from S. mutans backbone of lipoteichoic acid (WICKEN & KNOX strain BHT and several other strains of this bacterium showed in all cases a rapid 1971, MARKHAM et ai. 1975). Sheep erythrocytes coated with the lipoteichoic acid preparations adsorption to hydroxyapatite. A typical from 5. mutans were agglutinated by the antiexperiment is illustrated in Fig. I. The

ADSORPTION OF LIPOTEICHOIC ACID

389

adsorption isotherms (Fig. 1) show that 1 mg of untreated hydroxyapatite was able to take up a maximum of 14.2 ng (3,600 c.p.m.) of the cell-extracted lipoteichoic acid preparation, which was approximately 66% of the available radioactive polymers in the test system. Slightly ,A C less was adsorbed when the hydroxyO u apatite was pre-treated with saliva. The presence of 10 parts/10* of fluoride (NaF) in the liquid phase while adsorption was I occurring produced a marked reduction in the amount of teichoic acid taken up. The lipoteichoic acid adsorbed to the hydroxyapatite could be completely •I desorbed by the addition of 100 mM 4 5 phosphate buffer, pH 6.5; the presence of CPM ) 1 mM phosphate buffer during the initial adsorption did not significantly infiuence Fig, I. Adsorption of'H-lipoteichoic acid ('Hthe binding of lipoteichoic acid to the LTA) from S. mutans strain BHT tO' hydroxyhydroxyapatite. The extracellular lipo- apatite (HA). The curs'es show adsorption of teichoic acid preparation from other radioactive lipoteichoic acid to:: A, untreated strains exhibited very similar adsorption hydroxyapatite; B, saliva-treated hydroxyapatite; and C, hydroxyapatite in the presence isotherms and properties. of 10 parts/10' fluoride. Experimental conditions described in Material and Methods.

DiBcussion

The adsorption isotherm in Fig. 1 is a Brunauer type 1 and indicates the formation of a monomolecular layer of adsorbate, and a high affinity between

(WICKEN & KMOX 1975). This pheno-

menon may, however, be of minor importance in the adsorption of Grampositive bacteria to teeth, because the lipid adsorbant and adsorbate (ADAMSON part of the molecule would be more 1960). In the present study the affinity of intimately associated with the cell lipoteichoic acid for hydroxyapatite was membrane whereas the phosphate groups apparently strong because a concentration would be exposed at the cell surface. of less than 1 ^xM of this polymer The inhibiting effect of the anions, (assuming a molecular weight for lipid- phosphate and fluoride, on adsorption of free teichoic acid between 3,000-12,000; lipoteichoic acid indicates that the KNOX & WiCKENi973) can bind in the phosphate groups of this polymer interact presence of 1 mM phc-sphate. The with calcium ions on the surface of the observation that a maximum of only 14 ng hydroxyapatite (BERNARDI, GIRO SC of available lipoteichoic acid bound to GAILLARD 1972, ROLLA & MELSEN 1975a). ! mg hydroxyapatite could be due to the Fig. 2 shows a diagrammatic representaformation of non-adsorbing micelles at tion of the proposed binding - of lipoligh concentrations of the polymer teichoic acid to the calcium moiety of oecause of its amphipathic nature hydroxyapatite. The slight inhibition by

390 HYDROXYAPATITE/'^

CIARDI, R O L L A , BOWEN AND REILLY LIPOTEICHOIC ACID

ca*--o-P=O

ca* • - 0 - P = 0

Fig. 2. Diagrammatic representation of the proposed interaction between hydroxyapatite and lipoteichoic acid.

saliva might, in part, be due to the presence of acidic proteins (RoLLA 1976) that also bind to calcium ions of hydroxyapatite. The influence of fluoride on the adsorption of lipoteichoic acid to the hydroxyapatite (Fig. 1) supports the concept that this may be a valid model to study the interaction between the tooth surface and Gram-positive bacteria, because the results of recent clinical experiments have shown that fluoride reduces the bacterial colonization on teeth (LOESCHE, SYED, MURRAY & MELLBERG

significant amount of this iipoteichoic add released by S. mutans appears to be associated with insoluble polysaccharide synthesized by these microorganisms (MELVAER et al. 1974). Such a highly charged, relatively stable high molecular weight complex in the extracellular matrix could act as an ionic diffusion barrier in the dental plaque. Once the negatively charged phosphate groups of the polymer are saturated with the abundantly available calcium ions from saliva and/or demineralization of the tooth enamel, a positively charged barrier could exist, preventing further diffusion of positive ions from the vicinity of the tooth surface. This could result in a high hydrogen ion concentration at the tooth surface and account for the high cariogenic potential of 5. mutans. Clearly the observations reported here could have significant implications for the development of a vaccine against dental caries. Antibody against the lipoteichoic acid polysaccharide complex might prevent or reduce colonization of the tooth surface by microorganisms or affect the diffusion properties of plaque and thus reduce its cariogenicity. Acknowledgments — The authors gratefully acknowledge tlie technical assistance of Ms. LYNN KEMP and Mr. SHELDON GROVE.

1973, TiNANOFF, BRADY & GROSS 1976).

The obsen'ation that basic substances such as bis-biguaides, quaternar)' ammonium compounds and amines inhibit or reduce plaque formation (RoLLA & MELSEN 1975b, ROLLA 1976) further supports this concept becau.se all these substances could interact with the surface-bound teichoic acid of the bacteria and thus inhibit adsorption to dental enamel. The extracellular lipoteichoic acid was found to have very similar properties to the preparation extracted from the cells. A

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ADSORPTION OF LIPOTEICHOIC ACID

391

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G. Rolla Dental Faculty 71. Geitmyrsveien N-Osk 4 Norway