Interaction of salivary alpha-amylase and amylase-binding-protein A (AbpA) of Streptococcus gordonii with glucosyltransferase of S. gordonii and Streptococcus mutans

Abstract

Background: Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation by Streptococcus gordonii and Streptococcus mutans. The alpha-amylase-binding protein A (AbpA) of S. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs of S. gordonii and S. mutans.

Results: The addition of salivary alpha-amylase to culture supernatants of S. gordonii precipitated a protein complex containing amylase, AbpA, amylase-binding protein B (AbpB), and the glucosyltransferase produced by S. gordonii (Gtf-G). rAbpA was expressed from an inducible plasmid, purified from Escherichia coli and characterized. Purified rAbpA, along with purified amylase, interacted with and precipitated Gtfs from culture supernatants of both S. gordonii and S. mutans. The presence of amylase and/or rAbpA increased both the sucrase and transferase component activities of S. mutans Gtf-B. Enzyme-linked immunosorbent assay (ELISA) using anti-Gtf-B antibody verified the interaction of rAbpA and amylase with Gtf-B. A S. gordonii abpA-deficient mutant showed greater biofilm growth under static conditions than wild-type in the presence of sucrose. Interestingly, biofilm formation by every strain was inhibited in the presence of saliva.

Conclusion: The results suggest that an extracellular protein network of AbpA-amylase-Gtf may influence the ecology of oral biofilms, likely during initial phases of colonization.

Figure 1

SDS-PAGE analysis of amylase precipitates. Panel A. Precipitates from 18 h culture supernatants, were boiled in sample buffer. The samples containing 3.5 μg of protein were run on 12% SDS-PAGE, followed by staining with SYPRO red in 7.5% acetic acid solution. Lane 1, supernatant of wild type cells (WT); lane 2, supernatant of AbpA-deficient strain (ST); lane 3, supernatant of AbpB-deficient strain (SE); lane 4, molecular mass standard in kDa (M). Panel B. Precipitates from supernatants containing 1.4 μg of protein were used for Gtf-G activity assay. Samples were run on a 12% gel and stained for GTF activity. Lane 1, supernatant from wild type culture (WT); lane 2, supernatant from AbpA-deficient cells (ST); lane 3, supernatant from AbpB-deficient cells (SE). Panel C. Precipitates from supernatants containing 1.4 mg of total protein were used for Western Blot using polyclonal anti-AbpA antibody. Lane1, 2, and 3, supernatants from wild type (WT), AbpA-deficient (ST), and AbpB-deficient cells (SE), respectively.

Figure 2

Gtf enzyme activity assay. Overnight culture supernatant of strains in question, containing 3 μg total protein were used to precipitate Gtfs. To it, amylase and/or rAbpA was added to a final concentration of 50 μg/ml each. After 2 h at room temperature, precipitates were collected by centrifugation and resuspended in sample buffer and Gtf activity evaluated on SDS-polyacrylamide gel. Panel A. Lanes: 1, 2, and 3, supernatant from abpA^- strain (ST); lanes 4,5, and 6, supernatant from Gtf-deficient strain. Panel B. Lanes 1, 2, and 3, supernatant from S. mutans. Panel C. SDS-PAGE gel of precipitates from supernatants stained with SYPRO red. Lanes 1, 2, and 3, supernatants from AbpA-deficient strains; lanes 4, 5, and 6, supernatants from gtf-negative strains of S. gordonii; lanes 7, 8, and 9, supernatants from S. mutans.

Figure 3

Measurement of sucrase and transferase activities of GtfB. Purified GtfB enzyme was incubated at 37°C overnight with sucrose in presence or absence of amylase and/or rAbpA. The amount of glucose and fructose present in the reaction mixtures were measured using the F-kit. Panel A and B. Sucrase activity and transferase activity of GtfB in the presence of amylase and/or rAbpA, respectively. For statistical analysis, Gtf-B activities in the presence of amylase and/or rAbpA were compared with Gtf-B alone.

Figure 4

Binding of rAbpA and amylase with GtfB. For coating antigens onto wells of ELISA plate, 4, 2, 1, 0.5, and 0.25 μg of either amylase or rAbpA were used. To each well, 1 μg of ligand protein (Gtf-B) was added.

Figure 5

Growth and biofilm formation of wild-type, abp A and Δgtf mutants of S. gordonii. Bacteria were grown in BM or BM with 1% sucrose and/or 25% saliva. Growth and biofilm formation were measured under CO₂ enriched condition. All assays were performed eight times, and mean values and standard deviations are shown..