Abstract
Cariogenic Streptococcus mutans is known as a predominant etiological agent of dental caries due to its exceptional capacity to form biofilms. From strains of S. mutans isolated from dental plaque, we discovered, in the present study, a polyketide/nonribosomal peptide biosynthetic gene cluster, muf, which directly correlates with a strong biofilm-forming capability. We then identified the muf-associated bioactive product, mutanofactin-697, which contains a new molecular scaffold, along with its biosynthetic logic. Further mode-of-action studies revealed that mutanofactin-697 binds to S. mutans cells and also extracellular DNA, increases bacterial hydrophobicity, and promotes bacterial adhesion and subsequent biofilm formation. Our findings provided an example of a microbial secondary metabolite promoting biofilm formation via a physicochemical approach, highlighting the importance of secondary metabolism in mediating critical processes related to the development of dental caries.
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Data availability
The authors declare that all the data supporting the findings of the present study are available within the paper, the Supplementary Information and the Extended Data, and/or from the corresponding authors upon reasonable request. The MS raw data and searching results have been deposited to the ProteomeXchange Consortium via the PRIDE with the project accession no. PXD021641. The raw RNA-sequencing data have been deposited to the National Center for Biotechnology Information’s Sequence Read Archive database with the BioProject ID PRJNA667030.
References
Helöe, L. A. & Haugejorden, O. ‘The rise and fall’ of dental caries: some global aspects of dental caries epidemiology. Comm. Dent. Oral. Epidemiol. 9, 294–299 (1981).
Marsh, P. D. Are dental diseases examples of ecological catastrophes? Microbiology 149, 279–294 (2003).
Pitts, N. B. et al. Dental caries. Nat. Rev. Dis. Prim. 3, 17030 (2017).
Petersen, P. E., Bourgeois, D., Ogawa, H., Estupinan-Day, S. & Ndiaye, C. The global burden of oral diseases and risks to oral health. Bull. World Health Organ. 83, 661–669 (2005).
Listl, S., Galloway, J., Mossey, P. A. & Marcenes, W. Global economic impact of dental diseases. J. Dent. Res. 94, 1355–1361 (2015).
Kuramitsu, H. K., He, X., Lux, R., Anderson, M. H. & Shi, W. Interspecies interactions within oral microbial communities. Microbiol. Mol. Biol. Rev. 71, 653–670 (2007).
Lamont, R. J., Koo, H. & Hajishengallis, G. The oral microbiota: dynamic communities and host interactions. Nat. Rev. Microbiol. 16, 745–759 (2018).
Kolenbrander, P. E. Oral microbial communities: biofilms, interactions, and genetic systems. Annu. Rev. Microbiol. 54, 413–437 (2000).
Loesche, W. J. Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50, 353–380 (1986).
Lemos, J. A. et al. The biology of Streptococcus mutans. Microbiol. Spectr. 7, https://doi.org/10.1128/microbiolspec.gpp3-0051-2018 (2019).
Matsumoto-Nakano, M. Role of Streptococcus mutans surface proteins for biofilm formation. Jpn Dent. Sci. Rev. 54, 22–29 (2018).
Walsh, C. T. & Tang, Y. Natural Product Biosynthesis: Chemical Logic and Enzymatic Machinery (Royal Society of Chemistry, 2017).
Donia, M. S. & Fischbach, M. A. Small molecules from the human microbiota. Science 349, 1254766 (2015).
Liu, L., Hao, T., Xie, Z., Horsman, G. P. & Chen, Y. Genome mining unveils widespread natural product biosynthetic capacity in human oral microbe Streptococcus mutans. Sci. Rep. 6, 37479 (2016).
Aleti, G. 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 10, 00321–19 (2019).
Bushin, L. B., Clark, K. A., Pelczer, I. & Seyedsayamdost, M. R. Charting an unexplored streptococcal biosynthetic landscape reveals a unique peptide cyclization motif. J. Am. Chem. Soc. 140, 17674–17684 (2018).
Joyner, P. M. et al. Mutanobactin A from the human oral pathogen Streptococcus mutans is a cross-kingdom regulator of the yeast–mycelium transition. Org. Biomol. Chem. 8, 5486–5489 (2010).
Zvanych, R. et al. Systems biosynthesis of secondary metabolic pathways within the oral human microbiome member Streptococcus mutans. Mol. Biosyst. 11, 97–104 (2015).
Hao, T. et al. An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters. Nat. Commun. 10, 3665 (2019).
Tang, X. et al. Cariogenic Streptococcus mutans produces tetramic acid strain specific antibiotics that impair commensal colonization. ACS Infect. Dis. 6, 563–571 (2020).
Cornejo, O. E. et al. Evolutionary and population genomics of the cavity causing bacteria Streptococcus mutans. Mol. Biol. Evol. 30, 881–893 (2013).
Palmer, S. R. et al. Phenotypic heterogeneity of genomically-diverse isolates of Streptococcus mutans. PLoS ONE 8, e61358 (2013).
Hahnel, S., Rosentritt, M., Bürgers, R. & Handel, G. Adhesion of Streptococcus mutans NCTC 10449 to artificial teeth: an in-vitro study. J. Prosthet. Dent. 100, 309–315 (2008).
O’Toole, G., Kaplan, H. B. & Kolter, R. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54, 49–79 (2000).
Matsui, R. & Cvitkovitch, D. Acid tolerance mechanisms utilized by Streptococcus mutans. Future Microbiol. 5, 403–417 (2010).
Chau, N. P. T., Pandit, S., Jung, J.-E. & Jeon, J.-G. Evaluation of Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity. J. Dent. 42, 726–734 (2014).
van Loosdrecht, M. C. M., Lyklema, J., Norde, W. & Zehnder, A. J. B. Bacterial adhesion: a physicochemical approach. Microb. Ecol. 17, 1–15 (1989).
Absolom, D. R. et al. Surface thermodynamics of bacterial adhesion. Appl. Environ. Microbiol. 46, 90–97 (1983).
van Loosdrecht, M. C. M., Lyklema, J., Norde, W., Schraa, G. & Zehnder, A. J. B. The role of bacterial cell wall hydrophobicity in adhesion. Appl. Environ. Microbiol. 53, 1893–1897 (1987).
Vogler, E. A. Structure and reactivity of water at biomaterial surfaces. Adv. Colloid Interface Sci. 74, 69–117 (1998).
Van Oss, C. J. Interfacial Forces in Aqueous Media 2nd edn (Taylor & Francis, 2006).
Simões, L., Simões, M. & Vieira, M. Adhesion and biofilm formation on polystyrene by drinking water-isolated bacteria. Anton. Leeuw. 98, 317–329 (2010).
Stachelhaus, T., Mootz, H. D. & Marahiel, M. A. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493–505 (1999).
Kraas, F. I., Helmetag, V., Wittmann, M., Strieker, M. & Marahiel, M. A. Functional dissection of surfactin synthetase initiation module reveals insights into the mechanism of lipoinitiation. Chem. Biol. 17, 872–880 (2010).
Chen, H., O’Connor, S., Cane, D. E. & Walsh, C. T. Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit. Chem. Biol. 8, 899–912 (2001).
Perlova, O. et al. Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus. Appl. Environ. Microbiol. 72, 7485–7494 (2006).
Piel, J. Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 27, 996–1047 (2010).
Kosol, S., Jenner, M., Lewandowski, J. R. & Challis, G. L. Protein-protein interactions in trans-AT polyketide synthases. Nat. Prod. Rep. 35, 1097–1109 (2018).
Du, L. & Lou, L. PKS and NRPS release mechanisms. Nat. Prod. Rep. 27, 255–278 (2010).
Bloudoff, K. & Schmeing, T. M. Structural and functional aspects of the nonribosomal peptide synthetase condensation domain superfamily: discovery, dissection and diversity. Biochim. Biophys. Acta 1865, 1587–1604 (2017).
Müller, S. et al. Biosynthesis of crocacin involves an unusual hydrolytic release domain showing similarity to condensation domains. Chem. Biol. 21, 855–865 (2014).
Heathcote, M. L., Staunton, J. & Leadlay, P. F. Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units. Chem. Biol. 8, 207–220 (2001).
Cuthbertson, L. & Nodwell, J. R. The TetR family of regulators. Microbiol. Mol. Biol. Rev. 77, 440–475 (2013).
El-Khoury, N. et al. Spatio-temporal evolution of sporulation in Bacillus thuringiensis biofilm. Front. Microbiol. 7, 1222 (2016).
Yuan, X. Z. et al. Adsorption of surfactants on a Pseudomonas aeruginosa strain and the effect on cell surface lypohydrophilic property. Appl. Microbiol. Biotechnol. 76, 1189–1198 (2007).
Liu, B., Ge, N., Peng, B. & Pan, S. Kinetic and isotherm studies on the adsorption of tenuazonic acid from fruit juice using inactivated LAB. J. Food Eng. 224, 45–52 (2018).
Whitchurch, C. B., Tolker-Nielsen, T., Ragas, P. C. & Mattick, J. S. Extracellular DNA required for bacterial biofilm formation. Science 295, 1487 (2002).
Das, T., Kutty, S. K., Kumar, N. & Manefield, M. Pyocyanin facilitates extracellular DNA binding to Pseudomonas aeruginosa influencing cell surface properties and aggregation. PLoS ONE 8, e58299 (2013).
Strevett, K. A. & Chen, G. Microbial surface thermodynamics and applications. Res. Microbiol. 154, 329–335 (2003).
Hobley, L. et al. BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm. Proc. Natl Acad. Sci. USA 110, 13600–13605 (2013).
Blin, K. et al. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47, W81–W87 (2019).
Lau, P. C., Sung, C. K., Lee, J. H., Morrison, D. A. & Cvitkovitch, D. G. PCR ligation mutagenesis in transformable streptococci: application and efficiency. J. Microbiol. Methods 49, 193–205 (2002).
Kremer, B. H. et al. Characterization of the sat operon in Streptococcus mutans: evidence for a role of Ffh in acid tolerance. J. Bacteriol. 183, 2543–2552 (2001).
Li, L., Stoeckert, C. J. & Roos, D. S. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 13, 2178–2189 (2003).
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549 (2018).
Korenevsky, A. & Beveridge, T. J. The surface physicochemistry and adhesiveness of Shewanella are affected by their surface polysaccharides. Microbiology 153, 1872–1883 (2007).
Moon, K., Lim, C., Kim, S. & Oh, D. C. Facile determination of the absolute configurations of alpha-hydroxy acids by chiral derivatization coupled with liquid chromatography-mass spectrometry analysis. J. Chromatogr. A 1272, 141–144 (2013).
Freundlich, H. Ueber die adsorption in loesungen. Z. Phys. Chem. 57, 385–470 (1907).
Kwan, Y. H. et al. Comparative proteomics on deep-sea amphipods after in situ copper exposure. Environ. Sci. Technol. 53, 13981–13991 (2019).
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Acknowledgements
This work was financially supported by: grants from the Chan Zuckerberg Biohub Investigator Program to W.Z.; National Key R&D Programs of China (grant nos. 2018YFA0903200: MOST19SC04 and MOST19SC06), the China Ocean Mineral Resources Research and Development Association grant (grant no. COMRRDA17SC01) and Research Grant Council of HKSAR (grant no. C6026-19-A) to P.-Y.Q.; the Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) to W.Z. and P.-Y.Q. (grant no. SMSEGL20SC01); and the National Institutes of Health (grant no. R01 DE13239) to R.A.B. We thank J. G. Pelton (QB3, UC, Berkeley) for assistance with NMR measurements, A. T. Iavarone (QB3, UC, Berkeley) for assistance with MS experiments and D. Schichnes (CNR Biological Imaging Facility) and Z. Hu (UC, Berkeley) for assistance with microscope experiments.
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Z.-R.L. performed the experiments. Z.-R.L. and Y.D. analyzed the NMR data. Z.-R.L. and J.S. performed the gene and proteomic analyses. Z.-R.L. and A.P. performed the contact angle measurements. L.Z. and R.A.B. assisted with S. mutans culturing and genetics. Z.-R.L. and W.Z. designed the study and wrote the manuscript, with input from R.M., R.A.B. and P.-Y.Q.
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Extended data
Extended Data Fig. 1 S. mutans clinical isolates with diverse biosynthetic gene clusters (BGCs).
a, Organizations of seven natural product BGCs, which are harbored in 17 selected S. mutans clinical strains. The structures of SNC1-465, mutanobactin, and mutanocyclin and reutericyclin A are shown, whose biosynthesis is linked with BGC1, BGC3, and BGC4, respectively. b, Distribution of BGCs amongst 17 S. mutans strains. c, A phylogenetic tree of 17 S. mutans strains with the annotation of their corresponding BGCs.
Extended Data Fig. 2 The effect of muf on low pH resistance and surface adhesion of Smu57.
a, The effect of pH on the growth of Smu57 and its Δmuf mutant. b, Images of early stage biofilms (6-h-old) formed by Smu57 and Δmuf mutant, which were measured to evaluate the adhesion ability of these strains. c, Quantifications of early stage biofilm (6-h-old) formation by Smu57 and its six mutants in biofilm medium by crystal violet biofilm assay (OD 575). Results are presented as mean ± s.d. Data are representative of n = 3 independent experiments. Significance was assessed using one-sided Student’s t-test. a,b, Scale bars, 1 cm.
Extended Data Fig. 3 Confocal laser scanning microscopy (CLSM) images of stained biofilms.
The biofilms formed by Smu57 and its Δmuf mutant were measured by CLSM after staining with a LIVE/DEAD BacLight Bacterial Viability Kits (SYTO-9 and propidium iodide). Green fluorescence represents viable bacteria and red fluorescence represents affected bacteria. The conventional anticaries agent chlorhexidine (5 μM) was used as a positive control. The experiment was repeated two independent times with reproducible findings. Scale bar, 20 µm.
Extended Data Fig. 4 LC–HRMS extracted ion chromatogram traces of the metabolic extracts of muf+ strains.
a, A comparison of LC–HRMS extracted ion chromatogram traces of the metabolic extracts of Smu57 (upper panel) and its Δmuf mutant (bottom panel). A 1-hour elution condition was employed. b, LC–HRMS extracted ion chromatogram traces of the metabolic extract from Smu57 (upper panel), and HPLC trace of the same sample monitored at UV λ = 330 nm (bottom panel). A 30-min elution condition was employed. c, LC–HRMS extracted ion chromatogram traces of the metabolic extract of Smu50. d, LC–HRMS extracted ion chromatogram traces of the metabolic extract of Smu56. a–d, EIC + = 459.23 ± 0.01, 542.21 ± 0.01, 540.23 ± 0.01, 608.22 ± 0.01, and 698.25 ± 0.01, which correspond to compounds 1, 2, 3, 4, and 5, respectively.
Extended Data Fig. 5 Conversion of purified 5 to 4.
a, A proposed mechanism for the conversion of 5 to 4, accompanying the release of l-lactic acid (6). b, LC–HRMS extracted ion chromatogram trace of the degraded 5. EIC + = 89.02 ± 0.01, 606.21 ± 0.01, and 696.24 ± 0.01, which correspond to compounds 6, 4, and 5, respectively. Negative ion mode, LC–MS measured accurate mass spectrum was recorded across the range 50–1000 m/z.
Extended Data Fig. 6 Effects of Cys on the 5 production in various culture media.
a, Measurement of 5 production of Smu57 in biofilm medium supplemented with Cys at different concentrations. LC–HRMS extracted ion chromatogram traces of the metabolic extracts of Smu57 under different culture conditions were shown. EIC + = 698.25 ± 0.01, which corresponds to 5. b,c, Screening the optimal culture condition for 5 production. (b) A comparison of the yield of 5 from brain heart infusion (BHI) medium, BHI + Phe (1 mg/mL), BHI + Cys (1 mg/mL), and BHI + Phe+Cys (1 mg/mL for both amino acids). (c) A comparison of the yield of 5 from BHI medium supplemented with Cys at different concentrations. Results are presented as mean ± s.d. Data are representative of n = 3 independent experiments. Values with different letters represent significant differences (P < 0.05) determined by one-way ANOVA with Duncan’s test.
Extended Data Fig. 7 Effects of disruption of muf genes on the production of mutanofactins.
a, Measurement of 5 production of Smu57 and its ΔmufB, ΔmufC, and ΔmufHIJ mutants in biofilm medium. LC–HRMS extracted ion chromatogram traces of the metabolic extracts of Smu57 and its three mutants were shown. EIC + = 698.25 ± 0.01, which corresponds to 5. b,c, A comparison of the LC–HRMS extracted ion chromatogram traces of the metabolic extracts of (b) Smu57 and (c) its ΔmufB mutant. Both strains were grown in BHI medium. EIC + = 459.23 ± 0.01, 542.21 ± 0.01, 540.23 ± 0.01, 608.22 ± 0.01, and 698.25 ± 0.01, which correspond to compounds 1, 2, 3, 4, and 5, respectively. d, A comparison of the relative abundances of all mutanofactins from extracts of Smu57 and its ΔmufB mutant, based on the extracted ion chromatograms. Results are presented as mean ± s.d. Data are representative of n = 3 independent experiments. Significance was assessed using one-sided Student’s t-test.
Extended Data Fig. 8 Effects of compounds 3 and 4 on biofilm-forming ability of Smu57 Δmuf.
a, Images of biofilms formed by Δmuf mutant with 3 or 4 complementation. Scale bar, 1 cm. b, Quantification of biofilm formation by crystal violet biofilm assay (OD 575). Results are presented as mean ± s.d. Data are representative of n = 3 independent experiments. Significance was assessed using one-sided Student’s t-test.
Extended Data Fig. 9 Quantification of biofilms formed by cocultures of wild-type muf– strains with Smu56.
a, Crystal violet biofilm assay (OD 575) was employed for biofilm quantification. b, Images of typical biofilms formed by different co-culture samples. Scale bar, 1 cm. c, Colony-forming units (CFUs) of indicated strains grown in co-culture biofilms, which are shown in a,b, were counted. This result shows that Smu56 coexisted with all selected wild-type muf– strains. Although biofilm formation is an important microbial competition factor, Smu56 formed strong biofilm via producing compound 5, which could be hijacked by its neighboring muf– S. mutans strains. a,c, Results are presented as mean ± s.d. Data are representative of n = 3 independent experiments. a, Significance was assessed using one-sided Student’s t-test.
Extended Data Fig. 10 The synergistic effects of eDNA with 5 on bacterial aggregation and biofilm formation.
a, The synergistic effect of eDNA with 5 on bacterial cell aggregation. Images of cell self-aggregation with different treatments. b, Effect of the addition of eDNA (0.2 μg/mL or 2 μg/mL) on biofilm-forming ability of Smu57 Δmuf in biofilm medium with the complementation of compound 5 (2 μg/mL). Results are presented as mean ± s.d. Data are representative of n = 3 independent experiments. Significance was assessed using one-sided Student’s t-test. a,b, Scale bars, 1 cm.
Supplementary information
Supplementary Information
Supplementary Tables 1–10, Figs. 1–19, Notes 1–3 and References.
Supplementary Dataset 1
A summary of quantitative proteomic analysis.
Supplementary Dataset 2
A summary of transcriptome analysis.
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Li, ZR., Sun, J., Du, Y. et al. Mutanofactin promotes adhesion and biofilm formation of cariogenic Streptococcus mutans. Nat Chem Biol 17, 576–584 (2021). https://doi.org/10.1038/s41589-021-00745-2
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DOI: https://doi.org/10.1038/s41589-021-00745-2
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