The influence of saliva on infection of the human mouth by mutans streptococci.

The relationship between oral implantation of Streptococcus mutans IB1600 (serotype c) and Streptococcus sobrinus OMZ65 (serotype g), the aggregating activity of saliva, and its influence on the adherence of these bacterial strains in vitro was examined in seven human subjects. All the saliva samples aggregated strain IB1600 but not strain OMZ65 cells. Whole saliva from subjects with low levels of infection by Strep. mutans aggregated strain IB1600 to a greater degree than did whole saliva from those who were readily infected. Whole saliva from subjects most resistant to infection supported the adsorption of the highest number of either strain IB1600 or OMZ65 to hydroxyapatite surfaces. Thus the ability of whole saliva to aggregate Strep. mutans may influence the ability of these microorganisms to infect the mouth.

Glucan-binding factor in saliva.

High-molecular-weight polymers of alpha-1,6-linked D-glucans are insoluble in alcohol solutions. Whole, but not parotid, saliva prevented the precipitation of D-glucans by 80% (vol/vol) ethanol, showing that the whole saliva contained a factor which complexed with the glucan to render it alcohol soluble. The glucan-binding factor was retained on a column of Sephacryl S-200 which had been preequilibrated with 80% ethanol. The factor was then eluted with water. Passive hemagglutination assays revealed that the glucan-binding factor could sensitize erythrocytes to agglutination with anti-poly(glycerolphosphate), suggesting that the active glucan-binding component with lipoteichoic acid. The glucan-solubilizing factor was resistant to heat (100 degrees C), proteases, sialidase, lysozyme, lactoperoxidase, trichloroacetic acid, and Triton X-100. When sucrose was added to saliva, a suspension of Streptococcus cricetus AHT, or a suspension of Streptococcus sanguis 10556, relatively large amounts of glucan-binding factor were released in a soluble form. In addition, penicillin G caused the release of the glucan-solubilizing component from a suspension of S. cricetus AHT. It is suggested that whole saliva contains a component, tentatively identified as lipoteichoic acid, which can complex with glucans in a relatively hydrophobic solvent. This type of complex formation may be important in the adhesion of oral streptococci to saliva-coated surfaces.

Altered bacterial aggregation and adherence associated with changes in rat parotid-gland salivary proteins induced in vivo by beta-adrenergic stimulation.

Reduced adherence and aggregation were associated with protein alterations in parotid saliva after chronic treatment with the beta-adrenergic agonist isoproterenol. In contrast, saliva from animals treated with the beta-antagonist, propranolol, did not cause such changes; the protein composition of this saliva was similar to that of controls. SDS-polyacrylamide gel electrophoresis of protein in saliva samples before and after they were mixed with 10 mg of spheroidal hydroxyapatite beads (HA), as well as protein adsorbed and recovered from the HA, showed that an acidic, proline-rich protein with a molecular weight of approx. 40,000 was the predominant protein adsorbed. This protein was significantly diminished in saliva from isoproterenol-treated rats. Proteins with molecular weights between 44,000 and 48,000 and unique to the saliva from isoproterenol-treated animals were also adsorbed to HA. Thus alterations in proline-rich proteins of parotid saliva may influence the adherence and aggregation of oral bacteria, two processes considered important for in-vivo colonization of oral surfaces.

Cell-to-cell interaction of Streptococcus sanguis and Propionibacterium acnes on saliva-coated hydroxyapatite.

Cell-to-cell interaction (coaggregation) between Propionibacterium acnes PK93 and Streptococcus sanguis DL1 was measured on saliva-coated hydroxyapatite beads (SHA) at bacterial concentrations between 1.3 X 10(6) and 6.7 X 10(8) cells per ml. Four hundredfold more DL1 than PK93 cells adhered to the saliva-coated beads, and the adherence of S. sanguis was proportional to cell input. SHA precoated with 3 X 10(8) DL1 cells bound 75 to 80% of available PK93 cells at all input amounts tested, up to an input of 8 X 10(7) cells. Adherence of PK93 to DL1-coated SHA approached saturation at an input of approximately 10(9) PK93 cells, when 1.5 X 10(8) bound. The coaggregation on SHA occurred either in buffer or saliva and was inhibited by N-acetylgalactosamine and by lactose; the attachment of DL1 to SHA was not inhibited by these sugars. S. sanguis 34 and heat-treated DL1 cells, neither of which form coaggregates with PK93, attached to SHA, but such cells did not bind PK93 cells. The findings of this study indicate that bacteria unable to attach to saliva-coated hydroxyapatite can indeed adhere to such a surface by strong lectin-mediated cell-to-cell interactions with bacteria already attached to the surface.

Role of sucrose in plaque formation.

Results are presented which support the concept that the bacterial enzyme glucosyltransferase (GTF) plays a crucial role in sucrose induced plaque formation. GTF was shown to adhere strongly to anionic, hydrophobic and polysaccharide solid materials, and to be able to produce glucans in the adsorbed state. It appears conceivable that GTF adsorb to teeth and produce glucans. Glucan chains on the surface of the bacteria and glucans on the tooth surfaces interact (pack) and form a strong binding mechanism. The rigid alpha 1,3 linked glucans produced by Streptococcus mutans are particularly suited for interaction of this kind. This mechanism could account for sucrose-induced binding of bacteria to enamel, pellicle covered enamel and preformed plaque. S. mutans would adhere particularly strongly to tooth surfaces in the presence of sucrose, according to this model.

Effect of antibody preparations on glucose uptake by a cariogenic Streptococcus.

The effect of antisera to whole cells or cell wall components on glucose uptake by S. mutans 6715 was examined. Early stationary phase 6715 cells were treated with test serum and incubated at 37 degrees C in the presence of 14C-glucose. Samples were removed at timed intervals and measured in a liquid scintillation counter for radioactive uptake. Antisera to both whole cells and components known to be present on the surface of the cells reduced glucose uptake relative to normal serum. It is suggested that inhibition of glucose uptake may be one mechanism by which a caries vaccine may operate.

The effect of xylitol on plaque metabolism.

Evidence is presented which shows that Strep. mitior and Strep. mutans (which are unable to metabolize xylitol) take yp xylitol and transfer it to xylitol phosphate. Dental plaque also takes up xylitol. In this case a xylitol/protein complex is formed in addition to xylitol phosphate and also some labelled components yet to be identified. It is suggested that accumulation of xylitol phosphate inside the cells may "poison" the bacteria and possibly explain the caries therapeutic effect of xylitol observed in some laboratories.

Effect of topical application of stannous fluoride, stannous chloride and stannous tartrate on rat caries.

Topical application of 10 mM aqueous solutions of stannous fluoride inhibited caries in rats to a higher degree than 20 mM sodium fluoride, although the difference was not statistically significant. Furthermore, stannous fluoride reduced the number of Strep. mutans in plaque significantly; stannous ions have an antibacterial effect. Stannous chloride and stannous tartrate did not reduce caries in the rats, probably because of the low concentrations of available stannous ions in these solutions at low pH. The high concentration of stannous ions in solutions of stannous fluoride is probably partly due to the reduced hydroxide formation resulting from the buffering effect of HF formed at low pH in this solution.

Identification of IgA, IgG, lysozyme, albumin, alpha-amylase and glucosyltransferase in the protein layer adsorbed to hydroxyapatite from whole saliva.

IgA, IgG, albumin, lysozyme, alpha-amylase and glucosyltransferase were identified in the saliva coat which forms on hydroxyapatite exposed to whole saliva. It is suggested that these proteins may be involved in binding microorganisms to saliva coated hydroxyapatite in model studies.

Adsorption of glucosyltransferase to saliva coated hydroxyapatite. Possible mechanism for sucrose dependent bacterial colonization of teeth.

Glucosyltransferase (GTF) adsorbed to hydroxyapatite and to saliva coated hydroxyapatite in vitro. Several proteins which are known to be present in the "pellicle" which forms on hydroxyapatite when this mineral is exposed to whole saliva were shown to stimulate or inhibit GTF. It is suggested that these proteins may interact with GTF and cause binding of the enzyme to saliva coated hydroxyapatite. A model is suggested where GTF adsorbed to tooth surfaces may induce binding of microorganisms to tooth surfaces.

Rapid quantitative determination of the effect of antiplaque agents and antisera on the growth, acid production, and adherence of Streptococcus mutans.

A rapid quantitative in vitro assay measured the effects of antiplaque agents and antiserum on growth and sucrose-mediated adherence of radio-labeled S. mutans 6715. Acid production was measured by change in pH. In this assay the primary effect of chlorhexidine and fluoride was bacteriostasis and inhibition of acid production, respectively; the primary effect of dextran, dextranase, and specific antiserum was inhibition of sucrose-mediated adherence.

Antigens of Streptococcus mutans implicated in virulence--production of antibodies.

The present studies assayed antibody activities in serum and saliva of animals immunized by different routes, with cells of S. mutans or cell-free preparations containing GTF, FTF, LTA and/or dextranase synthesized by S. mutans. The results show that the type of immunogenic preparation and the route of its administration can elicit different antibody response and may in part explain the disparity of results achieved by different investigators. The results further emphasize the need to use standardized preparations and carefully described protocols for vaccination.

Purification, resolution, and interaction of the glucosyltransferases of Streptococcus mutans 6715.

The extracellular glucosyltransferase produced by Streptococcus mutans 6715 was purified from culture supernatant fluids to a specific activity of 9.6 IU/mg of protein, with an overall recovery of 87%. The purified enzyme preparation, designated unfractionated enzyme (UFE), synthesized only water-insoluble glucans from sucrose during the initial stages of the reaction, although some water-soluble polymers accumulated after extended periods of incubation. It was free from measurable fructosyltransferase activity. The UFE preparation was resolved into two different catalytically active components by ethanol fractionation. One fraction (designated insoluble product enzyme [IPE]) synthesized water-insoluble glucans, whereas the other (designated soluble product enzyme [SPE]) produced primarily water-soluble glucans. The difference between the insoluble glucans made by the UFE preparation and those made by the IPE fraction appeared to be due to interaction of the SPE and IPE components in the UFE preparation. Addition of commercial dextrans or enzymatically prepared glucans to the glucosyltransferase assay altered the amounts of soluble and insoluble glucans synthesized by the UFE preparation. The molecular weights of the major enzymatically active proteins producing insoluble and soluble glucans were estimated by gel filtration chromatography to be 150,000 and 175,000, respectively.

Adsorption of Streptococcus mutans lipoteichoic acid to hydroxyapatite.

Lipoteichoic acid extracted from cells of S. mutans strain BHT exhibited a high affinity for hydroxyapatite. 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.

Multicomponent nature of the glucosyltransferase system of Streptococcus mutans.

The glucosyltransferases of S mutans 6715 were resolved into two major fractions. One fraction synthesized water-soluble glucans and the other made water-insoluble glucans. Each fraction was found by polyacrylamide gel electrophoresis to be composed of several catalytically active species, apparently glycoprotein in nature. Treatment of the glucosyltransferases with dextranase in the absence of sucrose caused an interconversion of enzyme forms concomitant with a time-dependent loss of enzyme activity, but did not appear to remove significant amounts of the carbohydrate associated with the enzymes. Comparison of enzyme activity patterns on polyacrylamide gels of the five different S mutans serotypes further emphasizes the complexity of the glucosyltransferase system from this group of microorganisms.