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. 2022 Jan 7:2:796140.
doi: 10.3389/froh.2021.796140. eCollection 2021.

Tetramic Acids Mutanocyclin and Reutericyclin A, Produced by Streptococcus mutans Strain B04Sm5 Modulate the Ecology of an in vitro Oral Biofilm

Carla Uranga  1 , Karen E Nelson  1 , Anna Edlund  1   2 , Jonathon L Baker  1   2
Affiliations

Tetramic Acids Mutanocyclin and Reutericyclin A, Produced by Streptococcus mutans Strain B04Sm5 Modulate the Ecology of an in vitro Oral Biofilm

Carla Uranga et al. Front Oral Health. .

Abstract

The human oral microbiome consists of diverse microbes actively communicating and interacting through a variety of biochemical mechanisms. Dental caries is a major public health issue caused by fermentable carbohydrate consumption that leads to dysbiosis of the oral microbiome. Streptococcus mutans is a known major contributor to caries pathogenesis, due to its exceptional ability to form biofilms in the presence of sucrose, as well as to its acidophilic lifestyle. S. mutans can also kill competing bacteria, which are typically health associated, through the production of bacteriocins and other small molecules. A subset of S. mutans strains encode the muc biosynthetic gene cluster (BGC), which was recently shown to produce the tetramic acids, mutanocyclin and reutericyclins A, B, and C. Reutericyclin A displayed strong antimicrobial activity and mutanocyclin appeared to be anti-inflammatory; however the effect of these compounds, and the carriage of muc by S. mutans, on the ecology of the oral microbiota is not known, and was examined here using a previously developed in vitro biofilm model derived from human saliva. While reutericyclin significantly inhibited in vitro biofilm formation and acid production at sub-nanomolar concentrations, mutanocyclin did not present any activity until the high micromolar range. 16S rRNA gene sequencing revealed that reutericyclin drastically altered the biofilm community composition, while mutanocyclin showed a more specific effect, reducing the relative abundance of cariogenic Limosilactobacillus fermentum. Mutanocyclin or reutericyclin produced by the S. mutans strains amended to the community did not appear to affect the community in the same way as the purified compounds, although the results were somewhat confounded by the differing growth rates of the S. mutans strains. Regardless of the strain added, the addition of S. mutans to the in vitro community significantly increased the abundance of S. mutans and Veillonella infantium, only. Overall, this study illustrates that reutericyclin A and mutanocyclin do impact the ecology of a complex in vitro oral biofilm; however, further research is needed to determine the extent to which the production of these compounds affects the virulence of S. mutans.

Keywords: Streptococcus mutans (S. mutans); biofilm; dental caries; oral microbiome; oral microbiota; tetramic acid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The Streptococcus mutans B04Sm5 muc biosynthetic gene cluster (BGC) produces reutericyclins (A–C) and mutanocyclin. Illustration showing the chemical structures of reutericyclins (A–C) and mutanocyclin. Reutericyclins are made by MucA-E and are then processed by the MucF acylase to yield mutanocyclin [16].
Figure 2
Figure 2
The effect of reutericyclin and mutanocyclin on biofilm formation and acid production on an in vitro oral community. (A,B) Crystal violet biofilm assay results showing the dose responses of 0–10 nM of reutericyclin (A) and 0–500 μM mutanocyclin (B) on salivary biofilm formation after 16 h. (C) Effects of 0.5 nM reutericyclin and 500 μM mutanocyclin on the pH of the oral microbial community as a function of time. Statistical significance was determined using the Kruskal-Wallis one-way ANOVA, with a post-hoc Dunn's test. Error bars represent SEM.
Figure 3
Figure 3
The effect of reutericyclin and mutanocyclin on an in vitro oral community. (A) Stacked bar plots of 16S rRNA gene (16S) sequencing analysis showing percent abundances of taxa across all conditions tested. C = control, M = mutanocyclin, R = reutericyclin, B = biofilm, P = planktonic. (B,C) The effect of reutericyclin and mutanocyclin on alpha diversity in an oral microbial community. Alpha diversity (Shannon metric) of 16S sequencing grouped by A: Growth phase and B: Compound. Statistical differences between groups was assessed using Kruskal-Wallis test. *highlights p < 0.05 and ***highlights p < 0.001. (D) PCA biplot showing beta diversity (Robust Aitchison metric) and feature leadings generated with the QIIME2 [22] DEICODE plugin [23]. Data points represent the indicated samples and vectors illustrate the feature loadings (i.e. taxa) driving the differences in ordination space. Statistical differences between replicates assessed using a pairwise PERMANOVA analysis, displayed in the inset. **highlights p < 0.01. (E,F) Taxa with significantly different abundances upon treatment with reutericyclin or mutanocyclin. Bar charts illustrating the log2 fold change of taxa that had false discovery rate (FDR)-corrected adjusted p-values < 0.05 (based on DESeq2) upon treatment with either 0.5 nM reutericyclin (E) or 500 μM mutanocyclin (F). (G–I) Taxa found in the biofilm versus the supernatant. Bar charts illustrating the log2 fold change of taxa that had false discovery rate (FDR)-corrected adjusted p-values < 0.05 (based on DESeq2) in the biofilm or in the supernatant (planktonic) in the negative control (G), or upon treatment with either 0.5 nM reutericyclin (H) or 500 μM mutanocyclin (I).
Figure 4
Figure 4
Reutericyclin and mutanocyclin affect microbial correlations in an oral microbial community. Spearman association networks of 16S sequencing of the oral microbial community treated with either 500 μM mutanocyclin (A) or 0.5 nM reutericyclin (B), compared to a negative control. Nodes represent the species with the highest variances. Node size represents counts, node color represents hierarchical clustering, red and green edges represent negative and positive associations, respectively, with edge thickness representing higher Spearman's ρ.
Figure 5
Figure 5
Addition of reutericyclin and mutanocyclin-producing S. mutans to an in vitro oral biofilm community. (A) Stacked bar plots illustrating the % relative abundance of the indicated taxa across the samples with the indicated S. mutans strains added at the indicated ratio to the in vitro oral community. The results of 16S are shown in the top set of bars (DNA-based), while mRNA sequencing results (based on MetaPhlAn3) are shown in the bottom set of bars. (B,C) PCA biplots showing beta diversity [using Robust Aitchison metric in (B) and Bray-Cutris in (C)]. Data points represent the indicated samples, and vectors illustrate the feature loadings (i.e., taxa) driving the differences in ordination space. The results of 16S sequencing are shown (B), while mRNA sequencing results (based on MetaPhlAn3) are shown (C). Statistical differences between the replicates assessed using a pairwise PERMANOVA analysis, displayed in the insets. *p < 0.05 and **p < 0.01.
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References

    1. Bowen WH, Burne RA, Wu H, Koo H. Oral Biofilms: Pathogens, matrix, and polymicrobial interactions in microenvironments. Trends Microbiol. (2018) 26:229–42. 10.1016/j.tim.2017.09.008 - DOI - PMC - PubMed
    1. Lamont RJ, Koo H, Hajishengallis G. The oral microbiota: dynamic communities and host interactions. Nat Rev Microbiol. (2018) 16:745–59. 10.1038/s41579-018-0089-x - DOI - PMC - PubMed
    1. Rosier BT, De Jager M, Zaura E, Krom BP. Historical and contemporary hypotheses on the development of oral diseases: are we there yet?. Front Cell Inf Imm. (2014) 4:92. 10.3389/fcimb.2014.00092 - DOI - PMC - PubMed
    1. Aleti GA, Baker JL, Tang X, Alvarez R, Dinis M, Tran NC, et al. . Identification of the bacterial biosynthetic gene clusters of the oral microbiome illuminates the unexplored social language of bacteria during health and disease. mBio. (2019) 10:e00321–e00319. 10.1128/mBio.00321-19 - DOI - PMC - PubMed
    1. Hujoel PP, Lingstrom P. Nutrition, dental caries and periodontal disease: a narrative review. J Clin Periodontol. (2017) 44:S79–84. 10.1111/jcpe.12672 - DOI - PubMed

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