Genetic Loci Associated With Fluoride Resistance in Streptococcus mutans

Abstract

The prolonged exposure of the cariogenic bacterial species Streptococcus mutans to high concentrations of fluoride leads to the development of fluoride resistance in this species. Previous studies confirmed the involvement of a mutation in a single chromosomal region in the occurrence of fluoride resistance. The involvement of multiple genomic mutations has not been verified. The aim of this study is to identify multiple genetic loci associated with fluoride resistance in S. mutans. The previously published whole genome sequences of two fluoride-resistant S. mutans strains (UA159-FR and C180-2FR) and their corresponding wild-type strains (UA159 and C180-2) were analyzed to locate shared chromosomal mutations in fluoride-resistant strains. Both fluoride-resistant strains were isolated in laboratory by culturing their mother strains in media with high concentrations of fluoride. The corresponding gene expression and enzyme activities were accordingly validated. Mutations were identified in two glycolytic enzymes, namely pyruvate kinase and enolase. Pyruvate kinase was deactivated in fluoride-resistant strain C180-2FR. Enolase was less inhibited by fluoride in fluoride-resistant strain UA159-FR than in its wild-type strain. Mutations in the promoter mutp constitutively increased the promoter activity and up-regulated the expression of the downstream fluoride antiporters in fluoride-resistant strains. Mutations in the intergenic region glpFp led to lower expression of glpF, encoding a glycerol uptake facilitator protein, in fluoride-resistant strains than in wild-type strains. Our results revealed that there is overlap of chromosomal regions with mutations among different fluoride-resistant S. mutans strains. They provide novel candidates for the study of the mechanisms of fluoride resistance.

Keywords: Streptococcus mutans; caries prevention; fluoride; glycolysis; resistance; single nucleotide polymorphism.

FIGURE 1

The identification of loci with mutations shared between Streptococcus mutans UA159-FR and C180-2FR. (A) Scheme for the comparison of the SNPs from the two fluoride-resistant S. mutans strains. The nucleotide sequence of loci with mutations shared in both fluoride-resistant S. mutans strains is shown for (B) mutp, (C) glpFp, and (D) pyk. WT, wild-type strain; FR, fluoride-resistant strain; SNP, single nucleotide polymorphism; ^∗chromosomal regions in which mutations were found in both fluoride-resistant strains. perA and perB encode fluoride antiporters. glpF encodes a glycerol uptake facilitator protein. pepX encodes an x-prolyl-dipeptidyl aminopeptidase. pyk encodes pyruvate kinase. The nucleotides shown in red in (B) to (D) indicate identified SNPs. In (D), the amino acid substitutions due to the SNPs in pyruvate kinase are also shown in red.

FIGURE 2

Representative growth of S. mutans UA159 and UA159-FR on BHI agar plates. NaF is supplemented at increasing concentrations: (A) without NaF; (B) 4 mM NaF; (C) 8 mM NaF; (D) 12 mM NaF; (E) 25 mM NaF. Overnight grown cultures were serially diluted 10¹-fold (D1) to 10⁵-fold (D5).

FIGURE 3

Relative fold change of gene expression between S. mutans UA159 and UA159-FR. Results from S. mutans UA159 and UA159-FR at late exponential phase are shown. Overall expression of each selected gene in UA159-FR relative to that in UA159 is presented as average fold change ± standard deviation. This experiment was repeated three times. All p-values were corrected for multiple testing. ^∗p < 0.05; ^∗∗p < 0.005; and ^∗∗∗p < 0.0005.

FIGURE 4

Residual activity of pyruvate kinase in S. mutans C180-2, C180-2FR, UA159, and UA159-FR after incubation with different concentrations of NaF. The activity of the control group which was incubated without NaF was set at 100%. BDL, below detection limit.

FIGURE 5

Residual enolase activity of S. mutans UA159 and UA159-FR after pre-incubation with different concentrations of NaF. The activity of the control group in which no NaF was added during pre-incubation was set at 100%. ^∗∗p < 0.01 and ^∗∗∗p < 0.001.