F0F1-ATPase Contributes to the Fluoride Tolerance and Cariogenicity of Streptococcus mutans

Abstract

The phenotypic traits of Streptococcus mutans, such as fluoride tolerance, are usually associated with genotypic alterations. The aim of this study was to identify adaptive mutations of S. mutans to gradient fluoride concentrations and possible relationships between the mutations and fluoride tolerance. We identified a highly resistant S. mutans strain (FR1000) with a novel single nucleotide polymorphism (SNP, -36G→T) in the promoter region of F₀F₁-ATPase gene cluster (SMU_1527-SMU_1534) resistant to 1,000 ppm fluoride using the whole-genome Illumina PE250 sequencing. Thus, a -36G→T F₀F₁-ATPase promoter mutation from the parental strain S. mutans UA159 was constructed and named UA159-T. qRT-PCR showed that the F₀F₁-ATPase gene expression of both FR1000 and UA159-T was up-regulated, and fluoride tolerance of UA159-T was significantly improved. Complementation of Dicyclohexylcarbodiimide (DCCD), a specific inhibitor of F₀F₁-ATPase, increased fluoride susceptibility of FR1000 and UA159-T. Intracellular fluoride concentrations of fluoride tolerance strains were higher compared to UA159 strain as demonstrated by ¹⁸F analysis. Further validation with rat caries models showed that UA159-T caused more severe caries lesions under fluoride exposure compared with its parental UA159 strain. Overall, the identified -36G→T mutation in the promoter region of F₀F₁-ATPase gene drastically contributed to the fluoride tolerance and enhanced cariogenicity of S. mutans. These findings provided new insights into the mechanism of microbial fluoride tolerance, and suggested F₀F₁-ATPase as a potential target for suppressing fluoride resistant strains.

Keywords: Streptococcus mutans; antimicrobials; caries; fluoride(s); genomics.

FIGURE 1

Organization of gene clusters of S. mutans and their relation to intergenic SNPs. (A) Intergenic SNP upstream of pepX and glpF gene in FR600/FR1000. (B) Intergenic SNP upstream of tpx gene in FR1000. The -10A→T promoter mutation occurred in FR1000. (C) Intergenic SNP eriC1a (SMU_1290c) and eriC1b (SMU_1289c) in FR600 and FR1000. The -13T→C promoter mutation occurred in FR600 and FR1000. Intergenic SNPs of SMU_1292c and SMU_1291c in FR600 and FR1000. The -44T→G promoter mutation occurred in FR600 and FR1000. (D) Intergenic SNPs upstream of atpHGFEDCBA in FR1000. The -36G→T promoter mutation occurred in FR600 and FR1000. Sequences of intergenic regions were given in the blue bar. Red letter indicates SNPs; Purple box indicates putative -10 element (Pribnow box, share conserved sequences TATAAT/ATTATA); Pink box indicates putative -35 element (share conserved sequences TTGACA/TGTCAA); Red box indicates translation start site (ATG/CAT) of the operon.

FIGURE 2

−36G→T point mutation in UA159-T caused up-regulated atpH gene expression and fluoride resistance. (A) Quantitative real-time PCR of atpH: One -36G→T point mutation in F₀F₁-ATPase promoter significantly up-regulated atpH gene expression. Overall expression compared to UA159 was presented as average fold change ± SD. The significance level (α) was set at 0.05 (One-way ANOVA, Dunnett’s T3, IBeleM SPSS Statistics 24). *** indicates P < 0.001. ns indicates no significant difference. N = 12 per group. (B) Effects of gradient-concentration fluoride treatment on S. mutans growth: One -36G→T point mutation in F₀F₁-ATPase promoter significantly increased fluoride resistance. 1:100 diluted overnight suspension of strains was incubated in 96-well culture plates with different concentrations of fluoride (3/4-fold serial) for 24 h. N = 12 per group and per fluoride concentration. (C) Effects of gradient-concentration fluoride treatment on Colony-Forming Units of S. mutans: FR1000 and UA159-T showed higher fluoride tolerance than UA159. Five microliter suspension of the UA159, FR1000, UA159-T from the 96-well culture plate of MIC assays (treated with 3/4-fold serial dilutions of fluoride for 24 h) was incubated on TSA for 2 days. Images were taken by a microscopy.

FIGURE 3

Effects of Dicyclohexylcarbodiimide (DCCD), a specific F₀F₁-ATPase inhibitor, on fluoride resistance of S. mutans. (A) Fluoride inhibitory assays with 200 μM DCCD: suppression of the F₀F₁-ATPases by DCCD increased fluoride susceptibility of the fluoride-resistant strains in fluoride environment. The significance level (α) was set at 0.05 (independent two-sample t-test, two-tailed test, IBM SPSS Statistics 24). *** indicates P < 0.001. ns indicates no significant difference. N = 8 per group. (B) Effects of gradient-concentration fluoride treatment with 200 μM DCCD on S. mutans growth (N = 12 per group and per fluoride concentration): 200 μM DCCD suppressed F₀F₁-ATPases, increased fluoride susceptibility of the fluoride-resistant strains, and reversed the resistance effects induced by F₀F₁-ATPase over-expression.

FIGURE 4

Measurement of relative intracellular fluoride concentration. (A) The relative intracellular fluoride concentration increased alongside the external fluoride concentration. (B) With the existence of 4,000 ppm external fluoride, relative intracellular fluoride concentrations of fluoride resistant strains (FR1000 and UA159-T) were higher than UA159. The significance level (α) was set at 0.05 (One-way ANOVA, Dunnett’s T3, IBM SPSS Statistics 24). N = 6 per group. ** indicates P < 0.01. *** indicates P < 0.001. ns indicates no significant difference.

FIGURE 5

Dental caries conditions and Keyes’ scores of rat caries models. (A) Stereomicroscopy images of dental caries on maxillary and mandibular bones of rats infected with UA159 and UA159-T. Black arrows point out different severity in the same site of the two groups. UA159-T strains led to severer dental caries than UA159. (B) Keyes’ scores of smooth surfaces and sulcal surfaces of two group (UA159 and UA159-T) and N = 6 per group: UA159-T strains achieved higher Keyes’ scores and led to severer dental caries than UA159. The significance level (α) was set at 0.05 (independent two-sample t-test, two-tailed test, IBM SPSS Statistics 24). * indicates P < 0.05. ** indicates P < 0.01. ns indicates no significant difference.