Arginine Improves pH Homeostasis via Metabolism and Microbiome Modulation

Abstract

Dental caries can be described as a dysbiosis of the oral microbial community, in which acidogenic, aciduric, and acid-adapted bacterial species promote a pathogenic environment, leading to demineralization. Alkali generation by oral microbes, specifically via arginine catabolic pathways, is an essential factor in maintaining plaque pH homeostasis. There is evidence that the use of arginine in dentifrices helps protect against caries. The aim of the current study was to investigate the mechanistic and ecological effect of arginine treatment on the oral microbiome and its regulation of pH dynamics, using an in vitro multispecies oral biofilm model that was previously shown to be highly reflective of the in vivo oral microbiome. Pooled saliva from 6 healthy subjects was used to generate overnight biofilms, reflecting early stages of biofilm maturation. First, we investigated the uptake of arginine by the cells of the biofilm as well as the metabolites generated. We next explored the effect of arginine on pH dynamics by pretreating biofilms with 75 mM arginine, followed by the addition of sucrose (15 mM) after 0, 6, 20, or 48 h. pH was measured at each time point and biofilms were collected for 16S sequencing and targeted arginine quantification, and supernatants were prepared for metabolomic analysis. Treatment with only sucrose led to a sustained pH drop from 7 to 4.5, while biofilms treated with sucrose after 6, 20, or 48 h of preincubation with arginine exhibited a recovery to higher pH. Arginine was detected within the cells of the biofilms, indicating active uptake, and arginine catabolites citrulline, ornithine, and putrescine were detected in supernatants, indicating active metabolism. Sequencing analysis revealed a shift in the microbial community structure in arginine-treated biofilms as well as increased species diversity. Overall, we show that arginine improved pH homeostasis through a remodeling of the oral microbial community.

Keywords: biofilms; dental caries; dental plaque; metabolomics; microbiota; oral cavity.

Figure 1.

Arginine uptake and utilization assays. (A) Arginine uptake. Biofilms were incubated in 75 mM arginine for treatment time indicated above before washing and collection of cell pellets for liquid chromatography–mass spectrometry targeted arginine assay. Negative controls were not incubated in arginine and were collected at the indicated time point. Error bars = SD. (B) Arginine utilization. Biofilms were incubated in 75 mM arginine for 2 h, then washed and collected at the indicated time point after arginine removal. Supernatants were analyzed with gas chromatography–mass spectrometry, and the levels of the main arginine metabolites are shown. Negative controls were collected at 6 h. Error bars = SD.

Figure 2.

Pretreatment with arginine prior to sucrose exposure protects against pH drop. Pooled saliva was inoculated into SHI media overnight in biofilm-inducing conditions (see Materials and Methods). Biofilms were then washed and incubated in chemically defined media (CDM) adjusted to pH 7, plus arginine and/or sucrose as indicated. pH was measured by pipetting 20 μL onto color-changing pH strips. Adding sucrose 6 h after adding arginine resulted in an initial drop, followed by an increase in pH. Adding sucrose at 20 h or 48 h prevented the drop completely. Arg = L-arginine (75 mM); Suc = sucrose (15 mM).

Figure 3.

Arginine and metabolite quantification. Biofilms were collected at each time point and centrifuged. Pellets were analyzed with targeted liquid chromatography–mass spectrometry for arginine. Supernatants were analyzed with gas chromatography–mass spectrometry to detect and quantify metabolites. Left y-axes represent metabolite levels in supernatants; right y-axes represent the concentration of arginine in pellets. (A) Levels of select arginine-related metabolites and concentration of arginine in biofilms treated with 75 mM arginine at Time 0 and collected at 6 h, 20 h, and 54 h. (B) Levels of select arginine-related metabolites and concentration of arginine in differentially treated biofilms collected at 54 h. Arg = L-arginine (75 mM); Suc = sucrose (15 mM). Experiments were performed in duplicate. Error bars = SD.

Figure 4.

Relative abundance of (A) phylum-level, (B) genus-level, and (C) species-level taxa detected in each biofilm. Experiments were performed in duplicate, and relative abundances were averaged for each taxon detected. Arg = L-arginine (75 mM); Suc = sucrose (15 mM).

Figure 5.

Diversity analyses. (A) Beta diversity was calculated by unweighted UniFrac, and the distances were plotted with principal coordinate analysis (PCoA). The colors of each sample correspond to the colors on the bar graph in (B), and samples of the same color represent replicate experiments. Biofilms in which protection from the sucrose-induced pH drop occurred clustered separately from the biofilms in which pH protection did not occur; this correlates with the amount of time the biofilms were incubated in arginine prior to sucrose exposure. (B) The alpha diversity measure Chao1 was calculated at a defined sequence depth of 35,000 for all samples. Values for duplicate samples were averaged, and the error bars represent the standard deviation. “Initial inoculum” refers to the pooled saliva used to inoculate SHI medium to generate the initial biofilms, and “Time 0” refers to the overnight biofilms prior to the addition of arginine or sucrose.