Mead M. McCabe

Purification of dextran-binding protein from cariogenic Streptococcus mutans

Abstract An extracellular protein produced by Streptococcus mutans was purified to electrophoretic homogeneity by affinity chromatography on Sephadex G50 followed by gel filtration. The protein is devoid of both dextransucrase and dextranase activity but binds dextran and therefore probably is implicated in the adherence of S. mutans cells to the host tooth surface. The presence of the dextran-binding protein may be a determinant of the pathogenicity of such cariogenic micro-organisms.

The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R.

Soluble dextransucrase activity(ies) was eluted with a solution of clinical dextran from the insoluble dextran--cell complex produced by Streptococcus mutans K1-R grown in the presence of sucrose. Studies of the dextran acceptor-reaction of the soluble enzyme-preparation indicate that it is highly specific for dextran of high molecular weight. Increased dextran synthesis in the presence of dextran acceptor and the apparent inhibition of this stimulation by higher concentrations of dextran result from product modification rather than a direct effect on the level of enzyme activity. The results demonstrate that the potentially water-insoluble structure synthesized by dextransucrase on exogenous, soluble dextran acts as a more-efficient acceptor than the soluble dextran. The role of the acceptor reaction in the biosynthesis of complex dextrans is discussed.

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

Abstract An inhibitor of Streptococcus , mutans endodextranase was detected in proteins prepared from batch cultures of S. , mutans strains representing serotypes a through g . Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of S. , mutans known to produce this enzyme.

Multiple Forms of Dextran-Binding Proteins from Streptococcus Mutans

We have isolated a series of five proteins which appear to possess characteristic individual capacities for synthesizing dextrans and binding dextrans. Our suggestion that these proteins comprise an isozyme-like distribution of lectin and enzyme activities is, of course, very speculative and remains to be rigourously confirmed. However, the very identification of these several dextran binding proteins provides a biochemical basis to explain numerous observations suggesting that more than one mechanism for dextran binding is possessed by S. mutans (for instance: 24-27), especially the observations with mutants (24). These proteins probably are the molecular determinants of host infection by S. mutans and may prove to be potent immunogens for use in a vaccine. The presence of a dextran-binding lectin in S. mutans implicates this bacterial lectin in the earliest stage of infection: Attachment to host tissues. The multiplicity of proteins possessing characteristic dextran-synthesizing and dextran-binding capacities indicates the complexity of the adherence mechanisms evolved in S. mutans. Experiments with other bacteria (10-12, 28) suggest that bacterial lectins, in concert with host tissue carbohydrates, may be the molecular mediators of host recognition and subsequent initial attachment of bacterial cells to host tissues in non-pathogenic as well as pathogenic bacteria.

Esterase polymorphisms in the skipjack tuna, Katsuwonus pelamis

Abstract 1. 1. Five groups of esterases, identified by their migration rates, substrate specificities and inhibitor sensitivities, were resolved by starch gel electrophoresis. 2. 2. Electrophoretic variations of three esterases are described and genetic schemes are proposed to explain the variations of two of them. 3. 3. Esterase III is assumed to be a dimer. A single locus ( e - III ) with four alleles is proposed to account for the four observed phenotypes. 4. 4. Each isozyme of esterase Ib appears to be either a monomer, or a polymer of the same sub-unit. A single locus ( e - Ib ) with seven alleles, one of them a null allele, is proposed to explain the observed variations.

Multiple forms of 6-phosphogluconate dehydrogenase and alpha-glycerophosphate dehydrogenase in the skipjack tuna, Katsuwonus pelamis

Abstract 1. 1. Electrophoretic variations of 6-phosphogluconate dehydrogenase (6-PGD) in Katsuwonus pelamis appear to result from allelic differences at a single locus, pgd. 2. 2. The electrophoretic patterns of alpha-glycerophosphate dehydrogenase (GPD) appear to be influenced by both allelic differences at a single locus, gpd, and the consisent occurence of artefact bands. 3. 3. These two enzymes may not be genetically polymorphic.

An enzyme from Streptococcus mutans forms branches on dextran in the absence of sucrose

An enzyme in glucosyltransferase preparations from Streptococcus mutans catalyzed the transfer of [14C]glucopyranoside from purified isomaltosaccharides, each containing [14C]glucopyranoside at its non-reducing terminus, to acceptor dextran, in the absence of sucrose. Half of the radioactivity present in the resulting [14C]dextrans was resistant to hydrolysis by amylo-1,6-glucosidase. Treatment of the [14C]dextrans with endodextranase resulted in extensive hydrolysis and produced [14C]-labeled limit oligosaccharides containing branch sites. Acetolysis of the [14C]-labeled limit oligosaccharides yielded [14C]nigerose, thus indicating the formation of branch sites on dextran in the absence of sucrose. The enzyme catalyzing this reaction has not been identified but appears to be independent of the major extracellular glucosyltransferases of S. mutans.

Comments on adherence of Streptococcus mutans.

We do not agree with the cell adherence model proposed by Dr. Slade nor with the concept that cell-to-surface adherence and dextran-induced intercellular adherence in Streptococcus mutans are separate and dissociable traits. Our data indicate that cellto-surface and intercellular adherence are induced by similar mechanisms involving the same glucan receptor sites on the cell surface and differing only in the physical nature of the glucan bond, the relative strengths of the cell-glucan interactions, and the requirements for divalent cations. Thus, cell-to-surface adherence occurs when numerous cell-surface glucan receptors bind to adherent branched insoluble glucans, forming a highly stable cell-glucan complex, whereas intercellular adherence is mediated by the binding of the same glucan receptors to soluble glucans such as linear dextran and requires the formation of a stabilizing intercellular calcium bridge.1 Dr. Hutton Slades model for cell-to-surface adherence suggests that cell-bound glucosyltransferase (GTF) serves as both the means by which cells synthesize adherent glucans and as the only cell-surface binding site for these adherent glucans. This model is not compromised by Kuramitsus observation that GTF-free heat-killed S mutans cells will bind to a preformed layer of insoluble glucan deposited on a glass surface, using cell-free GTF preparations,2 since the model suggests that the cells bind to residual cellfree GTF that is already bound to the glucan layer. The model also suggests that dextran (linear a-1,6-glucan)-induced intercellular adherence is mediated by a separate dextran-specific binding site on the cell surface. The observation of mutant strains that readily agglutinate in the presence of sucrose, dextran, or soluble glucan but are not able to form adherent microbial deposits in vitro supports this suggestion.3