Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1

Abstract

Coaggregation between Streptococcus sanguis HI and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregationdefective mutant of S. sanguis Hi revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.Most bacteria isolated from the human oral cavity possess the ability to participate in intergeneric coaggregation. Coaggregation is characterized by a highly specific binding between stable surface components found on two different bacterial types. Intergeneric coaggregation is thought to play an important role in the formation of dental plaque deposits (11; P. E. Kolenbrander, Crit. Rev. Microbiol., in press). Streptococcus sanguis is one of the earliest colonizers of the clean tooth surface and is found in significant numbers in dental plaque (4,26 coaggregation-inhibiting polysaccharide (CIP) antigen from S. sanguis 34 has been isolated and characterized (18). This polysaccharide consists of rhamnose, glucose, galactose, and N-acetylgalactosamine in a hexasaccharide repeating unit (17). The polysaccharide inhibits coaggregation, contains saccharide components that by themselves are effective inhibitors of the interaction, and is a major cell surface antigen (18).To characterize the carbohydrate receptor from a different coaggregating pair, S. sanguis Hi and ...

Adhere today, here tomorrow: oral bacterial adherence

“…Coaggregation of Treponema denticola and P. gingivalis (15) correlates with the observation that T. denticola was never detected in periodontally affected sites unless that site was also inhabited by P. gingivalis (43 (26). Galactosides appear to be the sugar moiety most commonly recognized by oral bacterial lectins (26), although other sugars, such as rhamnose, have also been implicated (3,46 (Fig. 1); its specificity for galactoside-containing receptors suggests that the adhesin functions in a similar manner (16).…”

Microbial Biofilms: from Ecology to Molecular Genetics

“…For example, bacteria isolated from certain parts of the oral cavity tend to coaggregate with other bacteria isolated from the same location (e.g., bacteria isolated from the tongue coaggregate best with other tongue-localized bacteria [109]), suggesting a direct spatial organization in the formation of oral biofilms. Finally, coaggregation can be interrupted by the addition of certain sugars such as lactose and galactose, suggesting specific receptor-ligand interactions (29,137,265).…”

“…The receptor partners of the adhesins are believed to be cell wall-associated polysaccharides (possibly lipoproteins) which have been identified in a number of organisms, including S. sanguis (29) and Streptococcus oralis (160). These receptors are linear cell wall polysaccharides typically containing characteristic repeating units, including N-acetylglucosamine (1-3, 34, 35, 209; reviewed in reference 135).…”

Probing of Microbial Biofilm Communities for Coadhesion Partners

“…Using this assay, unidentified bacterial receptor-adhesin interactions, other than the ones based on recognition of RPS by type 2 actinomyces adhesins, could also be elucidated which have long been postulated to exist (34)(35)(36)(37)(38)(39). In broader sense, the coadhesion assay may be used to isolate and identify coadhesion partners from any type of mixed biofilm communities occurring in the human organism or elsewhere in nature.…”