Essential Roles of the sppRA Fructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression by Streptococcus mutans

Abstract

The dental caries pathogen Streptococcus mutans can ferment a variety of sugars to produce organic acids. Exposure of S. mutans to certain nonmetabolizable carbohydrates, such as xylitol, impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress in S. mutans was demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here, we studied an operon in S. mutans, sppRA, which was highly expressed in the fruK mutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg²⁺ and Mn²⁺ but inhibited by NaF. SppR, a DeoR family regulator, repressed the expression of the sppRA operon to minimum levels in the absence of the fructose-derived metabolite F-1-P and likely also F-6-P. The accumulation of F-1-P, as a result of growth on fructose, not only induced sppA expression, but it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression of sppA, via a plasmid or by deleting sppR, greatly alleviated fructose-induced stress in a fruK mutant, enhanced resistance to xylitol, and reversed the effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show that S. mutans is capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.IMPORTANCEStreptococcus mutans is a major etiologic agent for dental caries, primarily due to its ability to form biofilms on the tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon in S. mutans that regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways that fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.

Keywords: biofilm; fructose metabolism; sugar-phosphate phosphohydrolase; sugar-phosphate stress; xylitol.

FIG 1

Overexpression of sppA enhances growth of a fruK mutant on fructose. (A) Relative sppA mRNA levels measured in UA159/pIB184, UA159/pIB508, FruK-13/pIB184, FruK-13/pIB508, sppR/pBGE mutant, and sppR/pBGE-sppR mutant. Cultures were prepared using BHI medium and harvested at the exponential phase. Quantitative reverse transcription-PCR (qRT-PCR) was carried out using the total RNA samples, and the relative abundance of sppA was determined using the ΔΔC_q method. The error bars denote the standard deviations. Student’s t test was performed to determine the statistical significance of the data, with asterisks denoting a P value of <0.05. (B) FruK-13/pIB508 (red) and FruK-13/pIB184 (blue) were cultivated in BHI and then diluted into TV medium containing 10 mM fructose. The cultures were overlaid with mineral oil, and the OD₆₀₀ was measured every 30 min in a Bioscreen C. The results represent the average from at least three biological replicates.

FIG 2

In vitro characterization of SppA as a hexose-phosphate:phosphohydrolase. The majority of the reactions (except those in panels B and C) were performed at 37°C in a basic buffer composed of 50 mM PIPES (pH 6.5), 5 mM MgCl₂, and 1 mM MnCl₂. Release of phosphate was measured using a Malachite green reagent (MGR), and the results were divided by time and amounts of enzyme (A and D) or normalized for relative activities (B and C). (A) The indicated sugar-phosphates (10 mM) was incubated with His-SppA (0.08 µM for F-1-P, 0.67 µM for the rest), with release of phosphate being monitored over a period of 10 min. (B) F-1-P (0.1 mM) was incubated for 5 min with 0.33 µM His-SppA and the specified amounts of metal ions. (C) F-1-P (0.67 mM) was incubated with 0.04 µM His-SppA for 130 s at the indicated pH values (see Materials and Methods for details). (D) Increasing concentrations of F-1-P and F-6-P were each mixed with His-SppA, and phosphate release was monitored over defined periods of time (see Materials and Methods for details).

FIG 3

Transcription of the spp operon is regulated by SppR, fructose derivatives, and the PTS. The PsppR::cat fusion was integrated into the chromosome of the wild type (A) or various mutants (B) to monitor the expression of the operon. All strains were first cultivated to mid-exponential phase (OD₆₀₀, 0.3 to 0.4) in a TV medium with 10 mM glucose and then 25 or 50 mM the specified carbohydrates (A) or 50 mM glucose or fructose (B) was added, the cultures were incubated for 1 h, and cells were harvested for CAT assays. Error bars denote standard deviations. Asterisks indicate statistically significant difference (by Student’s t test, P < 0.05) in comparison with glucose at the same concentrations (A) or with the wild type (WT) treated with the same carbohydrates (B).

FIG 4

In vitro interactions of SppR with the spp promoter region and fructose phosphates. EMSA was performed using SppR and a biotin-labeled probe containing the promoter region of sppR. SppR (0 to 125 nM) was incubated first with various sugar-phosphates for 10 min and then with 0.2 nM DNA probe for 20 min before the mixture was resolved on a nondenaturing polyacrylamide gel.

FIG 5

Overexpression of SppA enhances resistance to xylitol. Growth phenotypes of various strains were monitored over time in a Bioscreen C (A and B) or by measuring the final yield (OD₆₀₀) after overnight incubation (C). Strains were cultivated first on BHI to exponential phase and then diluted using TV medium containing 0.5% glucose in addition to 1% xylitol (A and B) or the amounts of xylitol specified on the x axis (C).

FIG 6

Fructose metabolism alters biofilm development by S. mutans. BM containing 2 mM sucrose and 18 mM glucose (BMGS) or 18 mM fructose (BMFS) was used for biofilm development. (A) Wild-type UA159 and its isogenic levD and fruI mutants were each incubated for 24 h, and the amounts of biofilms formed were visualized and quantified by crystal violet (C.V.) staining. (B) Biofilms formed by UA159 were treated with a LIVE/DEAD vitality stain and visualized under a confocal laser scanning microscope, with green indicating live cells and red indicating dead cells. (C) eDNA released from 48-h biofilms formed by strains UA159/pIB184 and UA159/pIB508 were quantified using a DNA dye (Sytox green) and a standard curve. (D) The results were normalized using total CFU from each sample. Error bars represent standard deviations. Statistical analysis was performed using Student’s t test, with asterisks denoting P values: *, <0.05; **, <0.005; and ***, <0.0005. NS, nonsignificant; Glc, glucose; Fru, fructose.

FIG 7

HAD family phosphatases contribute to sugar-phosphate tolerance. (A) S. mutans UA159 and its mutants deficient in putative phosphohydrolases. (B) UA159, a lacD mutant, and lacD derivatives containing plasmid pIB428 that overexpresses SMU.428 or the vector pIB184. Bacterial cultures were each prepared in BHI and then diluted into TV base medium supplemented with 0.5% glucose and various amounts of xylitol (A) or 0.5% sorbitol and various amounts of galactose (B). After 17 to 20 h of incubation at 37°C, the OD₆₀₀ of the cultures was recorded. The results are the averages of at least three independent cultures, with error bars denoting the standard deviations.