Relationship between cell-bound dextransucrase and the agglutination of Streptococcus mutans

Lysophosphatidylcholine (LPC) and other phosphoglycerides stimulated waterinsoluble and water-soluble glucan production by the Streptococcus mutans 6715 dextransucrase (EC 2.4.1.5). LPC stimulated crude extracellular dextransucrase 1.7-fold, the water-insoluble glucan-producing a form of the enzyme 6.5-fold, the water-soluble glucan-producing ,B form of the enzyme 2.1-fold, and the cellassociated dextransucrase 2.0-fold. Kinetic studies demonstrated that LPC did not change the Km for sucrose of a or ,B but increased the maximum velocity of the enzymes. The Km for LPC of the a enzyme was 10' M. LPC from various sources and synthetic preparations of lauroyl-LPC, myristoyl-LPC, and palmitoyl-LPC all stimulated glucan formation. Portions of phosphoglyceride molecules including fatty acids, phosphatidic acid, glycerophosphoric acid, glycerophosphorylcholine, and choline, when tested individually or in combinations, did not enhance dextransucrase activity. The increased rates of glucan production caused by LPC and primer dextran were additive. Enzyme incubated with LPC before addition of sucrose was stimulated by dextran primer, and, conversely, enzyme treated with dextran was stimulated by addition of LPC with the sucrose substrate. Thus, dextransucrase can be activated by binding of intact phosphoglyceride molecules to a site on the enzyme that is distinct from either the glucosyl donor or glucosyl acceptor (primer) binding sites. Interactions between the S. mutans dextransucrase and amphipathic phosphoglycerides may explain properties of this enzyme which contribute to the cariogenicity of S. mutans. M sodium acetate, pH 5.5) at room temperature for 450 on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

mentioning

confidence: 49%

Streptococcus mutans Dextransucrase: Stimulation of Glucan Formation by Phosphoglycerides

Harlander

Schachtele

1978

“…Studies by Germaine and Schachtele (5) and the results of affinity chromatography (11,12,15) confirmed the ability of GTF to bind glucans. Other studies, however, indicated that cells depleted of GTF activity still agglutinated in the presence of glucans (14), thus suggesting that the GTF active site is not involved in glucan-mediated aggregation. An alternate candidate for the cell surface glucan-binding site is the glucan-binding protein (11), which recently was shown to possess exclusive specificity for 1,6-ot-D-glucans (E. C. Landale and M. M. Mc-Cabe, Abstr.…”

Section: Discussionmentioning

confidence: 92%

Purification and characterization of a primer-independent glucosyltransferase from Streptococcus mutans 6715-13 mutant 27

McCabe¹

1985

Self Cite

Affinity chromatography on Sephadex G-50 and subsequent ion-exchange chromatography on Trisacryl-M-DEAE were used to purify the glucosyltransferase (GTF) enzymes produced by mutant 27 of Streptococcus mutans 6715-13. Complete separation of three types of GTF, including a primer-independent GTF capable of synthesizing a slightly branched, water-soluble glucan (GTF-S), was obtained. The characteristics of this primer-independent GTF-S were compared with those of the normally occurring primer-dependent GTF-S. The Km for sucrose was easily obtained for each enzyme (10(-2) M), but the Km for dextran could only be determined for the primer-dependent GTF-S (5 X 10(-7) M for clinical dextran of molecular weight 60,000 to 90,000). The primer-independent GTF-S did not respond catalytically to the presence of either clinical dextran or the highly branched, water-soluble glucan produced by primer-dependent GTF-S, although it was capable of binding these polysaccharides at a noncatalytic site and of responding to the low-molecular-weight acceptor 1-O-methyl-alpha-D-glucopyranoside. The water-soluble glucan product of primer-independent GTF-S was a superior priming glucan for primer-dependent GTF enzymes as compared with the glucan product of primer-dependent GTF-S. The presence of primer-independent GTF-S in reaction mixtures stimulated glucan synthesis by primer-dependent GTF-S and by GTF synthesizing water-insoluble glucan by at least 10-fold, whereas the presence of similar amounts of primer-dependent GTF-S had no effect on synthesis by GTF synthesizing water-insoluble glucan. Primer-independent GTF-S appears to be a potent source of priming glucan for the primer-dependent GTF enzymes. Its possession of a noncatalytic binding site for glucan, the first observed for the GTF of S. mutans, suggests that it may also serve as a glucan receptor on the S. mutans cell surface.

“…Because previous results have suggested that receptors for branched glucans may be relatively heat stable (15,22), it is tempting to speculate that these molecules may play a role in converting extracellular GTF activity to the cell-associated form. In addition, glucans produced by cell-associated GTF activity would be heat stable.…”

Section: Mutansmentioning

confidence: 99%

Interaction of glucosyltransferase with the cell surface of Streptococcus mutans

Kuramitsu¹,

Ingersoll²

1978

The partially purified glucosyltransferase (GTF) fraction synthesizing primarily water-insoluble glucans, GTF-A, and the homogeneous fraction synthesizing water-soluble glucans, GTF-B, were utilized to assess the binding of GTF activity to the cell surface of Streptococcus mutans GS-5. Growth of the cells in either Todd-Hewitt broth or a chemically defined medium did not appear to affect the ability of the cells to bind either enzyme fraction. Heat inactivation of the cells did not singificantly reduce the interaction of the enzymes with the cells. Cell surface glucan molecules appear to be involved in GTF binding to the cells because: (i) dextranase or alpha-1,3-glucanase treatment of the cells markedly reduced enzyme binding; (ii) the inclusion of soluble dextrans in the binding assays reduced both GTF-A and GTF-B binding to the cells; and (iii) pretreatment of the cells or the GTF-B fraction with soluble dextrans before binding significantly reduced enzyme binding to the cells. In addition, enzyme binding appears to require a cell surface protein component because Pronase, but not trypsin, treatment of cells reduced enzyme binding. Furthermore, the removal of a portion of the cell surface GTF-glucan complex with 3 N NaCl appears to provide additional binding sites for the enzymes. These results are interpreted in terms of the mechanism of the conversion of extracellular GTF to the cell-associated form.