Cariogenic Streptococcus mutans Produces Tetramic Acid Strain-Specific Antibiotics That Impair Commensal Colonization

Abstract

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

Keywords: antibacterial bioactivity; biosynthesis; oral microbiome; reutericyclin; small molecule.

Figure 1

Identification of an orphan gene cluster from S. mutans and its metabolites. (a) Mutanocyclin gene cluster (muc) annotation. (b) HPLC profile of extracts from wild-type (WT) S. mutans B04Sm5 (i), S. mutans B04Sm5/ΔmucD (ii), and S. mutans B04Sm5/ΔmucF (iii). (c) Structures of metabolites identified from S. mutans in this study, including reutericyclin A (1), reutericyclin B (2), reutericyclin C (3), and mutanocyclin (4).

Figure 2

Characterization of MucF as acylase. (a) HPLC profiles of extracts from E. coli BAP1/pEXT06 (mucA–D) (i) and E. coli BAP1/pEXT07 (mucA–D + mucF) (ii). (b) Key HMBC correlations of compound 5. (c) HPLC analysis of trans-2-decenoic acid standard (i) extracts from wild-type (WT) S. mutans B04Sm5 (ii) and S. mutans B04Sm5/ΔmucD (iii). (d) HPLC analysis of isolated 4 as a standard (i), compound 1 incubated with E. coli Rosetta2 (DE3)pLys/pET28a (empty vector) cell lysate for 60 min (ii), and compound 1 incubated with E. coli Rosetta2 (DE3)pLys/pEXT26 (carrying mucF) for 10 min (iii), 30 min (iv), and 60 min (v).

Scheme 1. Model for 1–4 Biosynthesis

C, condensation; A, adenylation; T, thiolation; KS, ketosynthase; TE, thioesterase.

Figure 3

S. mutans uses the chemicals synthesized by muc to inhibit the growth of the competing species. (a) Deferred-antagonism assay, performed as described in the Methods, to observe the inhibition of other species by S. mutans. Cultures of S. mutans UA159, B04Sm5, ΔmucD, and ΔmucF were spotted on BHI agar or BHI agar buffered to pH 7 and incubated overnight. Cultures of S. sanguinis, S. gordonii, or S. mitis were added to 5 mL of soft BHI agar or pH 7 BHI agar and used to overlay the plates containing the S. mutans strains. Where indicated, 8 μL of purified MucF acylase protein was added to the spot of S. mutans and incubated for 3 h prior to the overlay with the 2nd species. Zones of inhibition were measured 24 h after addition of the assay. (b–d) Bar graphs illustrating the zones of inhibition produced by the indicated strains and conditions. Error bars represent standard deviation, and asterisks denote the statistical significance between indicated pairs as determined by Tukey’s multiple-comparison test following a two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) (n = 4). (e) Growth kinetics of the ΔmucF strain is impaired. Graph illustrating the growth of UA159, B04Sm5, ΔmucD, and ΔmucF in BHI (n = 8).