Comparative analysis of Gtf isozyme production and diversity in isolates of Streptococcus mutans with different biofilm growth phenotypes

Abstract

Streptococcus mutans is the main pathogenic agent of dental caries. Glucosyltransferases (Gtfs) produced by these bacteria are important virulence factors because they catalyze the extracellular synthesis of glucans that are necessary for bacterial accumulation in the dental biofilm. The diversity of GtfB and GtfC isozymes was analyzed in 44 genotypes of S. mutans that showed a range of abilities to form biofilms in vitro. Several approaches were used to characterize these isozymes, including restriction fragment length polymorphism analysis of the gtfB and gtfC genes, zymographic analysis of the identified GtfB and GtfC genotypes, and quantitation of isozyme production in immunoblot experiments with specific monoclonal antibodies. A high diversity of gtf genes, patterns of enzymatic activity, and isozyme production was identified among the isolates tested. GtfC and, to a lesser extent, GtfB were produced in significantly higher amounts by strains that had high biofilm-forming ability than by strains with low biofilm-forming ability. Biofilm formation was independent of the GtfB and GtfC genotype. Atypical strains that showed an apparent single Gtf isozyme of intermediate size between GtfB and GtfC were also identified. The results indicate that various expression levels of GtfB and GtfC isozymes are associated with the ability of distinct S. mutans genotypes to grow as biofilms, strengthening the results of previous genetic and biochemical studies performed with laboratory strains. These studies also emphasize the need to identify factors that control gtf gene expression.

FIG. 1.

Chromosome locus of the GS5 strain containing the gtfB and gtfC genes (GenBank accession number M17361). (A) Primer pairs used for specific amplification of gtfB and gtfC genes are represented as black and shaded arrows, respectively. The restriction sites of the 5.2-kb gtfB and 4.3-kb gtfC amplicons are represented by the dashed lines; within them, the size of fragments are indicated. (B) PCR products obtained with the GTFBF-GTFBR (lane 1) and GTFCF-GTFCR (lane 2) primer pairs. In 18 isolates, efficient amplification of the gtfC genes was not obtained. Instead, PCRs yielded two weaker products as shown in lane 2b. Thus, RFLP analysis of gtfC could not be performed with these 18 strains. ORF, open reading frame.

FIG. 2.

(A) Ten distinct RFLP classes of the gtfB amplicon (a to j) were detected by BsrI digestion among the clinical strains analyzed. (B) Patterns of RFLP identified by SspI digestion of gtfC amplicons (k to o).

FIG. 3.

Representative patterns of Gtf isozyme production and activities analyzed in clinical isolates representative of the gtfB and gtfC genotypes identified by RFLP. GtfB and GtfC were electrophoretically separated on quadruplicate SDS-6% PAGE gels and then analyzed for protein by Coomassie staining, for glucan production after incubation of gels with sucrose, and immunologically for GtfB and GtfC in Western blots developed with MAb P72 or MAb P32, respectively. Strains are indicated above each lane. RFLP classes of gtfB and gtfC amplicons are shown beneath each lane. Note that strains 4SM1 and 3SN1 presented a glucan-synthesized band of intermediate size between the typical GtfB- and GtfC-related glucan bands. Strains 5ST1 and 11SSST2, representative of a single S. mutans amplitype, did not display detectable levels of GtfB.

FIG. 4.

Distribution of Gtf isozyme production and biofilm growth in 44 S. mutans amplitypes. The relative amounts of Gtf produced were expressed as the intensities of the immunological dot blot reactions adjusted for the A₅₅₀ of the cultures tested. Biofilm growth was expressed as the A₅₇₅ of ethanol-eluted crystal violet of the stained biofilms. A significant positive association was observed between amounts of GtfB (A) and GtfC (B) produced and biofilm formation. No positive association was detected between amounts of GtfD and biofilm growth (C).