The well-coordinated linkage between acidogenicity and aciduricity via insoluble glucans on the surface of Streptococcus mutans

Abstract

Streptococcus mutans is considered the principal cariogenic bacterium for dental caries. Despite the recognition of their importance for cariogenesis, the possible coordination among S. mutans' main virulence factors, including glucan production, acidogenicity and aciduricity, has been less well studied. In the present study, using S. mutans strains with surface-displayed pH-sensitive pHluorin, we revealed sucrose availability- and Gtf functionality-dependent proton accumulation on S. mutans surface. Consistent with this, using a pH-sensitive dye, we demonstrated that both in vivo cell-produced and in vitro enzymatically synthesized insoluble glucans displayed proton-concentrating ability. Global transcriptomics revealed proton accumulation triggers the up-regulation of genes encoding functions involved in acid tolerance response in a glucan-dependent manner. Our data suggested that this proton enrichment around S. mutans could pre-condition the bacterium for acid-stress. Consistent with this hypothesis, we found S. mutans strains defective in glucan production were more acid sensitive. Our study revealed for the first time that insoluble glucans is likely an essential factor linking acidogenicity with aciduricity. The coordination of these key virulence factors could provide new insights on how S. mutans may have become a major cariogenic pathogen.

Figure 1. Percentage changes of fluorescence intensity in S. mutans wild-type and gtfBC-deficient strains upon switching to different pH values.

S. mutans strain O87 [O87] and its gtfBC-deficient strain [gtfBC/O87] were pre-grown in buffered pH7.5 medium and switched to the indicated pH values. The fluorescence signal reduction was calculated as the percentage of signal reduction relative to the initial signal at pH7.5. The plots show the average of triplicate samples, and the error bars correspond to the standard deviations. Student’s t-test was used to calculate the significance of the difference between the strain O87 supplemented with sucrose and that supplemented with glucose, and also between the gtfBC mutant and its parent strain. *Star indicates statistical significance between the value of indicated sample and that of strain O87 subjected to the same treatment (p < 0.05).

Figure 2. pHrodo Green STP Ester staining of S. mutans cells and in vitro prepared insoluble glucans.

(A) S. mutans UA 140 wildtype or gtfBC mutant cells pre-grown in the buffered (pH7.0) MDM supplemented with sucrose (20 mM) were harvested and stained with in pHrodo green dye in buffers with different pH (pH 5.5 and 7.0); and (B) in vitro prepared insoluble glucans were stained by pHrodo Green STP Ester in pH7 buffer.

Figure 3. Global transcriptome profiling and differentially expressed genes previously observed in Acid Tolerance Response (ATR) and sugar metabolism in WT and gtfBC mutant detected through RNAseq.

(A) Differentially expressed genes between pH 5.5 and pH 7.5 across the genome in WT (upper left panel) and gtfBC deficient stain (upper right panel). Expression ratios are log2 fold changes. Expressed genes represented by open circles are colored and sized based on the log₂ fold change in expression between WT at pH 5.5 and pH 7.5. Gene expression changes in the gtfBC mutant that show similar or opposing trends as the WT are then visible in the plot. (B) Differential expression of select key genes previously associated with ATR as well as sugar metabolism pathways and their log₂ fold changes. Red dashed vertical lines indicate significant fold change.

Figure 4. Acid killing assay of S. mutans wt and gtfBC strains pre-grown the presence or absence of sucrose.

Cells of S. mutans wild-type [WT] and gtfBC-deficient strain [gtfBC] were pre-grown in the buffered minimal medium (pH 7.5) supplemented with glucose or sucrose (20 mM). The cultures were then rapidly acidified to pH 2.8 using glycine solution, and incubated for 0, 0.5 h and 1 h, respectively. The survivors were recovered by plating on TH agar. Survival rate was calculated as the CFU mL⁻¹ at a given time divided by the CFU mL⁻¹ at time zero. Values marked with an asterisk are statistically significantly different between groups (P < 0.05, ANOVA comparison for all pairs using Tukey-Kramer HSD).

Figure 5. Illustration of qRT-PCR and transcriptomic data comparing gtfBC deletion and WT revealing insoluble glucan linking acidogenicity and aciduricity in S. mutans.

(A) S. mutans establishes low localized pH via its surface-associated glucans and that low surface pH induces protective mechanisms for coping with acid-stress. The normal adaptive Acid Tolerance Responses (ATR) genes in WT are not induced when glucans are missing. (B) Illustration of how the lack of acid tolerance is likely to impact the overall persistence and growth of S. mutans within the community and (C) a model of how the virulence properties could be linked.