Amino Sugars Enhance the Competitiveness of Beneficial Commensals with Streptococcus mutans through Multiple Mechanisms

Abstract

Biochemical and genetic aspects of the metabolism of the amino sugars N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) by commensal oral streptococci and the effects of these sugars on interspecies competition with the dental caries pathogen Streptococcus mutans were explored. Multiple S. mutans wild-type isolates displayed long lag phases when transferred from glucose-containing medium to medium with GlcNAc as the primary carbohydrate source, but commensal streptococci did not. Competition in liquid coculture or dual-species biofilms between S. mutans and Streptococcus gordonii showed that S. gordonii was particularly dominant when the primary carbohydrate was GlcN or GlcNAc. Transcriptional and enzymatic assays showed that the catabolic pathway for GlcNAc was less highly induced in S. mutans than in S. gordonii Exposure to H2O2, which is produced by S. gordonii and antagonizes the growth of S. mutans, led to reduced mRNA levels of nagA and nagB in S. mutans When the gene for the transcriptional regulatory NagR was deleted in S. gordonii, the strain produced constitutively high levels of nagA (GlcNAc-6-P deacetylase), nagB (GlcN-6-P deaminase), and glmS (GlcN-6-P synthase) mRNA. Similar to NagR of S. mutans (NagRSm), the S. gordonii NagR protein (NagRSg) could bind to consensus binding sites (dre) in the nagA, nagB, and glmS promoter regions of S. gordonii Notably, NagRSg binding was inhibited by GlcN-6-P, but G-6-P had no effect, unlike for NagRSm This study expands the understanding of amino sugar metabolism and NagR-dependent gene regulation in streptococci and highlights the potential for therapeutic applications of amino sugars to prevent dental caries.

Importance: Amino sugars are abundant in the biosphere, so the relative efficiency of particular bacteria in a given microbiota to metabolize these sources of carbon and nitrogen might have a profound impact on the ecology of the community. Our investigation reveals that several oral commensal bacteria have a much greater capacity to utilize amino sugars than the dental pathogen Streptococcus mutans and that the ability of the model commensal Streptococcus gordonii to compete against S. mutans is substantively enhanced by the presence of amino sugars commonly found in the oral cavity. The mechanisms underlying the greater capacity and competitive enhancements of the commensal are shown to depend on how the genes for the catabolic enzymes are regulated, the role of the allosteric modulators affecting such regulation, and the ability of amino sugars to enhance certain activities of the commensal that are antagonistic to S. mutans.

FIG 1

Growth curves of S. gordonii DL1, S. salivarius 57.I, and S. mutans UA159 on modified FMC medium containing 20 mM GlcNAc (A) or GlcN (B). Cultures were inoculated using exponentially growing cells supported by BHI, and the optical density (OD₆₀₀) was monitored using a Bioscreen C machine maintained at 37°C, with readings taken every 30 min.

FIG 2

Liquid coculture competition between S. mutans (S. m.) (UA159 Km-resistant derivative) and S. gordonii (S. g.) (DL1 Em-resistant derivative). The strains were each cultivated in TV supplemented with Glc or GlcNAc until exponential phase before being inoculated at a 1:1 ratio into fresh TV medium containing 20 mM Glc, GlcNAc, or GlcN. After 20 h of incubation, aliquots of the cultures were diluted and plated onto BHI agar plates containing antibiotics. The relative abundance of both strains at the start of the competition was similarly monitored (data not shown). Values within the bars show the proportions of the cultures constituted by S. mutans or S. gordonii, as indicated by dark or light shading, respectively. Error bars represent standard deviations.

FIG 3

Dual-species biofilms formed using S. mutans (S. m.) and S. gordonii (S. g.). Equal amounts of UA159-Km and DL1-Em cells were used to inoculate biofilm medium (BM) supported by 18 mM Glc and 2 mM sucrose (BMGS) and allowed to form a biofilm on a glass coverslip. After 24 h, the medium was replaced by BM supplemented with 20 mM Glc, GlcN, or GlcNAc, or fresh BMGS. After another 24 h of incubation, biofilms were harvested for CFU counting (A) and imaging by confocal laser scanning microscopy (B). Values within the bars in panel A show the percentage of the populations constituted by S. mutans or S. gordonii, as indicated by dark or light shading, respectively. Error bars represent standard deviations.

FIG 4

Growth phenotypes of the EI (ptsI) mutant of S. gordonii on TV-based agar plates containing Glc, GlcN, or GlcNAc. Also included are the wild-type parental strain DL1 and strains of S. mutans: wild-type UA159 and ptsI, nagA, and nagB mutants.

FIG 5

Measurements of NagA (A) and NagB (B) specific (sp) activities in cell lysates of S. mutans (wild-type UA159 and its nagA and nagB mutants) and S. gordonii DL1. The nagA and nagB mutants were cultivated only on Glc. Error bars represent standard deviations.

FIG 6

Quantitative real-time RT-PCR measurements of the message levels of nagA (A), nagB (B), and glmS (C) genes in S. gordonii wild-type DL1 and its nagR-null mutant. Error bars represent standard deviations.

FIG 7

Protein-DNA interactions assessed by EMSA and FP. A recombinant NagR (sg) was overexpressed as MBP-NagR in E. coli, purified and released by protease cleavage, and then used in an EMSA with biotin-labeled DNA fragments (2 fmol per reaction) containing promoter regions upstream of glmS, nagB, and nagA from S. gordonii (A), and a fluorescent polarization (FP) assay (B) against a 6-FAM-labeled probe containing consensus binding sequence of dre. CHO, carbohydrate.