Coaggregation reactions between actinomycete and streptococcal cells occurred frequently when human strains of Actinomyces viscosus or A. naeslundii were mixed with human isolates of Streptococcus sanguis or S. mitis, but were infrequent with other oral actinomycetes and streptococci. Two groups of actinomycetes and four groups of streptococci were defined by the patterns of their coaggregation reactions and by the ability of fl-linked galactosides (i.e., lactose) to reverse these reactions. Coaggregations occurred by one of the following three kinds of cell-cell interactions: (i) coaggregation that was blocked by heating the streptococcus but not the actinomycete and was not reversed by lactose; (ii) coaggregation that was blocked by heating the actinomycete but not the streptococcus and was reversed by lactose; and (iii) coaggregation that was blocked only by heating both cell types. The latter reaction was a combination of the first two since lactose reversed coaggregation between heated streptococci and unheated actinomycetes but did not reverse coaggregations between unheated streptococci and heated actinomycetes. Cells that could be heat inactivated also were inactivated by amino group acetylation or protease digestion, whereas cells that were unaffected by heat were not inactivated by these treatments. Coaggregation reactions of each kind were Ca2" dependent and insensitive to dextranase treatment. These findings are consistent with the hypothesis that human strains of A. viscosus and A. naeslundii coaggregate with strains of S. sanguis and S. mitis by a system of specific cell surface interactions between protein or glycoprotein receptors on one cell type and carbohydrates on the other type.

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Actinomyces viscosus T14V and Streptococcus sanguis 34 coaggregate by a mechanism which is not inhibited by 1 M NaCl, is dextran independent, requires calcium, is pH dependent with an optimum at pH 8.0 to 8.5, and appears to require the interaction of a protein or glycoprotein on A. viscosus with a carbohydrate on S. sanguis. The coaggregation is inhibited more than 80% by 0.01 M lactose, 0.02 M /l-methyl-D-galactoside, or 0.05 M D-galactose; inhibition of coaggregation was less than 10% in 0.1 M a-methyl-D-galactoside, melibiose, maltose, cellobiose, sucrose, and a number of monosaccharides. At very high concentrations of enzyme, protease from S. griseus destroyed the reactive site on A. viscosus but not on S. sanguis. Both were totally resistant to dextranase. Periodate (0.01 M; pH 4) inactivated both bacteria. The ability of S. sanguis to coaggregate with A. viscosus was not destroyed by phenol-water extraction at 65°C for 15 min. When the bacteria were cultured under specified conditions, the coaggregation was highly reproducible. Under the same conditions, T14AV, the avirulent mutant of A. viscosus T14V, did not coaggregate with S. sanguis 34. Electron microscopic studies of coaggregates, labeled immunochemically with antibody to A. viscosus, indicated that fibrils on A. viscosus may be involved in the coaggregation.

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4°C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously ( nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(al-3)Rha(frl-4)Glc(I1-6)GalftIl-6)GalNAc(P1-3)Gal(a1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing OiGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.Many of the specific adherence interactions between different bacterial species in dental plaque (15) appear to involve cell-associated lectins on one organism interacting with carbohydrates on another (7-9, 12, 17, 22, 23). One such interaction, lactose-sensitive coaggregation of Actinomyces viscosus T14V with Streptococcus sanguis 34, involves a lectin on A. viscosus T14V and a carbohydrate on S. sanguis 34. Studies have shown that this coaggregation is inhibited much more effectively by Gal,-OMe than by Gala-OMe and more so by Gal(,B1-3)GalNAc than by any other of several disaccharides tested (24-26). While such findings contribute to characterization of the A. viscosus T14V lectin, they provide little insight as to the structure(s) of lectin receptors on S. sanguis 34. We now describe the isolation and characterization of a coaggregation-inhibitory polysaccharide (CIP) from S. sanguis 34 cells that inhibits coaggregation of this organism with A. viscosus T14V and which presumably functions as the receptor on the streptococcal surface.

Coaggregation between Actinomyces viscosus T14V (T14V) and Streptococcus sanguis 34 (Ss34) depends upon specific reaction between lectin on T14V and carbohydrate on Ss34. Studies on coaggregation inhibition by sugars related to Dgalactose, ,B-galactosides, and amphipathic molecules revealed: (i) D-fucose, Dtalose approximately equal to D-galactose, which was 0.2 potency of lactose. No other hexoses or pentoses inhibited at 0.1 M. (ii) GalP(1->3)GalNAcaOCH2C6H5 was the most potent P-galactoside inhibitor; it had 20 times the potency of lactose.