Discovery of Novel Iminosugar Compounds Produced by Lactobacillus paragasseri MJM60645 and Their Anti-Biofilm Activity against Streptococcus mutans

Abstract

The oral cavity contains a number of microbes. They interact with each other and play an important role in human health. Among oral cariogenic microbes, Streptococcus mutans is recognized a major etiological bacteria of dental caries. Lactobacilli strains have been promoted as possible probiotic agents against S. mutans. However, their inhibitory mechanism has not been well elucidated yet. In the present study, two new compounds with strong antibiofilm activities were purified from the culture supernatant of Lactobacillus paragasseri MJM60645, which was isolated from the human oral cavity. These compounds showed strong inhibitory activities against S. mutans biofilm formation, with IC₅₀ (concentration at which 50% biofilm was inhibited) of 30.4 μM for compound 1 and 18.9 μM for compound 2. However, these compounds did not show bactericidal activities against S. mutans. Structure elucidation by nuclear magnetic resonance (NMR) and mass spectrometry showed that compound 1 was composed of two arabinofuranose iminosugars jointed with one glycerol and oleic acid, and compound 2 was composed of two arabinofuranose iminosugars jointed with one glycerol and nervonic acid. To the best of our knowledge, these structures were discovered for the first time in this study. Treatment of S. mutans with compound 1 strongly downregulated expression levels of genes related to biofilm formation, including gtfB, gtfC, gtfD, gbpB, brpA, spaP, ftf, and smu0630 without affecting the expression of comDE or relA. This study provides new insights into novel molecules produced by Lactobacillus to regulate the pathogenesis of S. mutans, facilitating a better understanding of the mechanism for interactions between Lactobacillus and S. mutans. IMPORTANCE In this study, we isolated lactic acid bacteria that inhibit streptococcal biofilm from the oral cavity of infants and identified two novel compounds from the supernatant of their culture broth. The two compounds are structurally similar, and both consist of iminosugars, glycerol, and unsaturated fatty acid. A search of the SciFinder database revealed that these structures are novel and were discovered for the first time in this study. Mechanism studies have shown that these compounds can inhibit the expression of biofilm synthesis-related genes. This is the first report that lactic acid bacteria inhibit streptococcal biofilms by small molecules with new chemical structures. This study not only expands the understanding of natural products derived from lactic acid bacteria but also provides a new paradigm for the understanding of the interaction of bacteria in the oral microbiota.

Keywords: Lactobacillus paragasseri; Streptococcus mutans; biofilm; iminosugar; oral microbiota.

FIG 1

Neighbor-joining tree based on the 16S rRNA gene sequences showing the phylogenetic relationship of strain MJM60645 and its related taxa. The accession numbers in parentheses were obtained from NCBI. The numbers at nodes indicate percentage levels of bootstrap support based on a neighbor-joining method of 1,000 replications. The evolutionary distances were computed using the Kimura two-parameter method. The scale bar indicates 0.005 substitutions per nucleotide position.

FIG 2

pH and thermal stability of crude extract of L. paragasseri MJM60645. (A) Biofilm formation in the presence of the crude extract of different pH values. Control was treated with water of different pH levels. (B) Biofilm formation after treated with crude extracts that were heated at different temperatures for 30 or 60 min or 121°C for 15 min. Control was not treated with the crude extract. The data are expressed as the means ± standard deviations from three biologically independent experiments. ****, P < 0.0001, indicating a significant difference compared with the control groups. Statistical analysis was performed with one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test.

FIG 3

Effect of crude extract on the number of viable S. mutans in planktonic and biofilm cells. (A) The total viable S. mutans (planktonic + biofilm) after treated with the crude extracts at different concentrations. *, P < 0.05 by Student’s two-tailed t test. (B) The cells embedded in the biofilm were compared to the total cells. The CFU values represent the means ± standard deviations from three independent experiments. The asterisks indicate the significant difference between control and extract-treated groups. *, P < 0.05; **, P < 0.01. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple-comparison test.

FIG 4

Structures of antibiofilm compounds produced by L. paragasseri MJM60645. (A) Structure of compound 1. (B) Structure of compound 2.

FIG 5

Determination of antibiofilm IC₅₀ of isolated compounds. (A, B) Dose-response curve of inhibition by compounds 1 (A) and 2 (B) on S. mutans biofilm formation. The data are presented as mean percentages of inhibition ± standard deviation for a total of five samples (pooled from three independent experiments in triplicate). The percentage of inhibition is relative to phosphate-buffered saline (PBS) control.

FIG 6

Effect of compounds 1 and 2 on early (A) and mature (B) biofilm formation. (A) S. mutans was treated with compounds 1 and 2 at the concentration of 60 and 36 μM, respectively, at the beginning of the assay. After incubation for 24 h, the biofilm was measured. (B) S. mutans was incubated in a brain heart infusion (BHI) medium containing 0.2% sucrose in a 96-well plate. After incubation for 24 h, the developed biofilm was treated with compounds 1 and 2 and incubated for a further 24 h. The biofilm was determined as described above. All measurements were performed in triplicates, and the mean values ± SD were calculated. ****, P < 0.0001. Statistically significant differences were calculated by one-way ANOVA with Dunnett’s multiple-comparison test.

FIG 7

Effect of compound 1 on the expression of biofilm-associated genes in S. mutans. S. mutans was treated with compound 1 at the concentration of 60 μM in a BHI medium containing 0.2% of sucrose and incubated anaerobically at 37°C for 24 h. Biofilm cells were harvested, and the expression of biofilm-associated genes was assessed by real-time quantitative PCR (qRT-PCR). The mRNA expression levels were calibrated using 16S rRNA. The data are expressed as the means ± standard deviations from three biologically independent experiments. The asterisks indicate that the gene expression between the control group (white bars) and the compound 1-treated group (black bars) was significantly different. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple-comparison test.