Role of Streptococcus gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm formation - PubMed (original) (raw)

Role of Streptococcus gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm formation

J D Rogers et al. Infect Immun. 2001 Nov.

Abstract

Interactions between bacteria and salivary components are thought to be important in the establishment and ecology of the oral microflora. alpha-Amylase, the predominant salivary enzyme in humans, binds to Streptococcus gordonii, a primary colonizer of the tooth. Previous studies have implicated this interaction in adhesion of the bacteria to salivary pellicles, catabolism of dietary starches, and biofilm formation. Amylase binding is mediated at least in part by the amylase-binding protein A (AbpA). To study the function of this protein, an erythromycin resistance determinant [erm(AM)] was inserted within the abpA gene of S. gordonii strains Challis and FAS4 by allelic exchange, resulting in abpA mutant strains Challis-E1 and FAS4-E1. Comparison of the wild-type and mutant strains did not reveal any significant differences in colony morphology, biochemical metabolic profiles, growth in complex or defined media, surface hydrophobicity, or coaggregation properties. Scatchard analysis of adhesion isotherms demonstrated that the wild-type strains adhered better to human parotid-saliva- and amylase-coated hydroxyapatite than did the AbpA mutants. In contrast, the mutant strains bound to whole-saliva-coated hydroxyapatite to a greater extent than did the wild-type strains. While the wild-type strains preincubated with purified salivary amylase grew well in defined medium with potato starch as the sole carbohydrate source, the AbpA mutants did not grow under the same conditions even after preincubation with amylase. In addition, the wild-type strain produced large microcolonies in a flow cell biofilm model, while the abpA mutant strains grew much more poorly and produced relatively small microcolonies. Taken together, these results suggest that AbpA of S. gordonii functions as an adhesin to amylase-coated hydroxyapatite, in salivary-amylase-mediated catabolism of dietary starches and in human saliva-supported biofilm formation by S. gordonii.

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Figures

FIG. 1

FIG. 1

PCR screening of abpA::erm(AM) recombinants of S. gordonii. Lane 1, abpA (544-bp) positive control amplified from Challis; lane 2, recombinant in S. gordonii Challis-E2 resulting from the duplication (single crossover) of abpA (544 bp) and insertion of abpA::erm(AM) (2.5 kb); lane 3, recombinant in S. gordonii Challis-E1 resulting from allelic exchange (double crossover) of abpA::erm(AM) (2.5-kb band only).

FIG. 2

FIG. 2

Binding of 125I-labeled amylase to S. gordonii strains Challis (●), Challis-E1 (○), FAS4 (▾), FAS4-E1 (▿), and S. sanguis 10556 (▪). Data points represent the mean values of duplicate samples from two experiments.

FIG. 3

FIG. 3

Comparison of culture supernatant levels of AbpA from wild-type and AbpA mutants of S. gordonii. An amylase ligand-binding assay was used. Lanes are loaded with equal amounts of protein (25 μg) from culture supernatants of S. gordonii Challis-E1 (lane 1), Challis (lane 2), FAS4 (lane 3), and FAS4-E1 (lane 4).

FIG. 4

FIG. 4

Binding isotherms for the adherence of S. gordonii strains to coated HAP. (a) Whole-saliva-coated HAP; (b) HPS-coated HAP; (c) glycosylated amylase-coated HAP; (d) nonglycosylated amylase-coated HAP. Data for the following S. gordonii strains are shown: Challis (●), Challis-E1 (○), FAS4 (▾), FAS4-E1 (▿), and S. sanguis 10556 (▪).

FIG. 5

FIG. 5

Scatchard plots for adherence of S. gordonii strains to coated HAP. (a) Whole-saliva-coated HAP; (b) HPS-coated HAP; (c) glycosylated amylase-coated HAP; (d) nonglycosylated amylase-coated HAP. Data for the following S. gordonii strains are shown: Challis (●), Challis-E1 (○), FAS4 (▾), FAS4-E1 (▿), and S. sanguis 10556 (▪).

FIG. 6

FIG. 6

Amylase-facilitated growth of amylase-binding and nonbinding streptococcal strains. Bacteria were grown in a defined medium with 2% potato starch as the primary carbon source, and growth was monitored spectrophotometrically at 600 nm. All cells were incubated with amylase prior to inoculation into the culture medium. Data for the following S. gordonii strains are shown: Challis (●), Challis-E1 (○), FAS4 (▾), FAS4-E1 (▿), and S. sanguis 10556 (▪). Data represent mean values of three experiments, each done in duplicate.

FIG. 7

FIG. 7

Live/Dead-stained biofilms of three random fields of view from the flow cell containing S. gordonii Challis (top row) and three random fields of view of the abpA mutant Challis-E1 (bottom row) after overnight growth on 25% saliva. Image dimensions are 250 by 250 μm. Maximum microcolony height is about 10 μm (wild type) and about 5 μm (mutant). The vast majority of cells stained green in the wild-type biofilm, but the majority of the cells were red to orange in the mutant biofilm.

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