Article | Published:

Identification of autoinducing thiodepsipeptides from staphylococci enabled by native chemical ligation

Abstract

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

Primary sequencing data are deposited at the National Centre for Biotechnology Information (NCBI GenBank). All other data generated and analysed during this study are available in the article and its Supplementary Information. Further details are available from the corresponding author on request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Ji, G., Beavis, R. C. & Novick, R. P. Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl Acad. Sci. USA 92, 12055–12059 (1995).

  2. 2.

    Novick, R. P. & Geisinger, E. Quorum sensing in staphylococci. Annu. Rev. Genet. 42, 541–564 (2008).

  3. 3.

    Thoendel, M., Kavanaugh, J. S., Flack, C. E. & Horswill, A. R. Peptide signaling in the staphylococci. Chem. Rev. 111, 117–151 (2011).

  4. 4.

    Wang, B. & Muir, T. W. Regulation of virulence in Staphylococcus aureus: molecular mechanisms and remaining puzzles. Cell Chem. Biol. 23, 214–224 (2016).

  5. 5.

    Ji, G., Beavis, R. & Novick, R. P. Bacterial interference caused by autoinducing peptide variants. Science 276, 2027–2030 (1997).

  6. 6.

    Otto, M., Süßmuth, R., Vuong, C., Jung, G. & Götz, F. Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives. FEBS Lett. 450, 257–262 (1999).

  7. 7.

    Mayville, P. et al. Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc. Natl Acad. Sci. USA 96, 1218–1223 (1999).

  8. 8.

    McDowell, P. et al. Structure, activity and evolution of the group I thiolactone peptide quorum-sensing system of Staphylococcus aureus. Mol. Microbiol. 41, 503–512 (2001).

  9. 9.

    Lyon, G. J., Mayville, P., Muir, T. W. & Novick, R. P. Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase AgrC. Proc. Natl Acad. Sci. USA 97, 13330–13335 (2000).

  10. 10.

    Lyon, G. J., Wright, J. S., Muir, T. W. & Novick, R. P. Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus. Biochemistry 41, 10095–10104 (2002).

  11. 11.

    George, E. A., Novick, R. P. & Muir, T. W. Cyclic peptide inhibitors of staphylococcal virulence prepared by Fmoc-based thiolactone peptide synthesis. J. Am. Chem. Soc. 130, 4914–4924 (2008).

  12. 12.

    Tal-Gan, Y., Stacy, D. M., Foegen, M. K., Koenig, D. W. & Blackwell, H. E. Highly potent inhibitors of quorum sensing in Staphylococcus aureus revealed through a systematic synthetic study of the group-III autoinducing peptide. J. Am. Chem. Soc. 135, 7869–7882 (2013).

  13. 13.

    Tal-Gan, Y., Stacy, D. M. & Blackwell, H. E. N-Methyl and peptoid scans of an autoinducing peptide reveal new structural features required for inhibition and activation of AgrC quorum sensing receptors in Staphylococcus aureus. Chem. Commun. 50, 3000–3003 (2014).

  14. 14.

    Johnson, J. G., Wang, B., Debelouchina, G. T., Novick, R. P. & Muir, T. W. Increasing AIP macrocycle size reveals key features of agr activation in Staphylococcus aureus. ChemBioChem 16, 1093–1100 (2015).

  15. 15.

    Tal-Gan, Y., Ivancic, M., Cornilescu, G., Yang, T. & Blackwell, H. E. Highly stable, amide-bridged autoinducing peptide analogues that strongly inhibit the AgrC quorum sensing receptor in Staphylococcus aureus. Angew. Chem. Int. Ed. 55, 8913–8917 (2016).

  16. 16.

    Hansen, A. M. et al. Lactam hybrid analogues of solonamide B and autoinducing peptides as potent S. aureus AgrC antagonists. Eur. J. Med. Chem. 152, 370–376 (2018).

  17. 17.

    Yang, T., Tal-Gan, Y., Paharik, A. E., Horswill, A. R. & Blackwell, H. E. Structure–function analyses of a Staphylococcus epidermidis autoinducing peptide reveals motifs critical for AgrC-type receptor modulation. ACS Chem. Biol. 11, 1982–1991 (2016).

  18. 18.

    Canovas, J. et al. Cross-talk between Staphylococcus aureus and other staphylococcal species via the agr quorum sensing system. Front. Microbiol. 7, 1733 (2016).

  19. 19.

    Gless, B. H. et al. Structure–activity relationship study based on autoinducing peptide (AIP) from dog pathogen S. schleiferi. Org. Lett. 19, 5276–5279 (2017).

  20. 20.

    Paharik, A. E. et al. Coagulase-negative staphylococcal strain prevents Staphylococcus aureus colonization and skin infection by blocking quorum sensing. Cell Host Microbe 22, 746–756 (2017).

  21. 21.

    Gordon, C. P., Olson, S. D., Lister, J. L., Kavanaugh, J. S. & Horswill, A. R. Truncated autoinducing peptides as antagonists of Staphylococcus lugdunensis quorum sensing. J. Med. Chem. 59, 8879–8888 (2016).

  22. 22.

    Otto, M., Süßmuth, R., Jung, G. & Götz, F. Structure of the pheromone peptide of the Staphylococcus epidermidis agr system. FEBS Lett. 424, 89–94 (1998).

  23. 23.

    Jarraud, S. et al. Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus. J. Bacteriol. 182, 6517–6522 (2000).

  24. 24.

    Kalkum, M., Lyon, G. J. & Chait, B. T. Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry. Proc. Natl Acad. Sci. USA 100, 2795–2800 (2003).

  25. 25.

    Olson, M. E. et al. Staphylococcus epidermidis agr quorum-sensing system: signal identification, cross talk, and importance in colonization. J. Bacteriol. 196, 3482–3493 (2014).

  26. 26.

    Todd, D. A. et al. Signal Biosynthesis Inhibition with Ambuic Acid as a Strategy To Target Antibiotic-Resistant Infections. Antimicrob. Agents Chemother. 61, e00263-17 (2017).

  27. 27.

    Tsuda, S., Yoshiya, T., Mochizuki, M. & Nishiuchi, Y. Synthesis of cysteine-rich peptides by native chemical ligation without use of exogenous thiols. Org. Lett. 17, 1806–1809 (2015).

  28. 28.

    Wang, B., Zhao, A., Novick, R. P. & Muir, T. W. Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence. Proc. Natl Acad. Sci. USA 112, 10679–10684 (2015).

  29. 29.

    Rink, H. Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin. Tetrahedron Lett. 28, 3787–3790 (1987).

  30. 30.

    Dufour, P. et al. High genetic variability of the agr locus in Staphylococcus species. J. Bacteriol. 184, 1180–1186 (2002).

  31. 31.

    Pyörälä, S. & Taponen, S. Coagulase-negative staphylococci—emerging mastitis pathogens. Vet. Microbiol. 134, 3–8 (2009).

  32. 32.

    Devriese, L. A., Hájek, V., Oeding, P., Meyer, S. A. & Schleifer, K. H. Staphylococcus hyicus (Sompolinsky 1953) comb. nov. and Staphylococcus hyicus subsp. chromogenes subsp. nov. Int. J. Syst. Evol. Microbiol. 28, 482–490 (1978).

  33. 33.

    Tong, S. Y. et al. Novel staphylococcal species that form part of a Staphylococcus aureus-related complex: the non-pigmented Staphylococcus argenteus sp. nov. and the non-human primate-associated Staphylococcus schweitzeri sp. nov. Int. J. Syst. Evol. Microbiol. 65, 15–22 (2015).

  34. 34.

    Novick, R. P., Ross, H. F., Figueiredo, A. M. S., Abramochkin, G. & Muir, T. W. Activation and inhibition of the staphylococcal AGR system. Science 287, 391 (2000).

  35. 35.

    Kamath, U., Singer, C. & Isenberg, H. D. Clinical significance of Staphylococcus warneri bacteremia. J. Clin. Microbiol. 30, 261–264 (1992).

  36. 36.

    Webster, J. A. et al. Identification of the Staphylococcus sciuri species group with EcoRI fragments containing rRNA sequences and description of Staphylococcus vitulus sp. nov. Int. J. Syst. Evol. Microbiol. 44, 454–460 (1994).

  37. 37.

    Nakatsuji, T. et al. Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis. Sci. Transl. Med. 9, eaah4680 (2017).

  38. 38.

    Barros, E. M., Ceotto, H., Bastos, M. C. F., dos Santos, K. R. N. & Giambiagi-deMarval, M. Staphylococcus haemolyticus as an important hospital pathogen and carrier of methicillin resistance genes. J. Clin. Microbiol. 50, 166–168 (2012).

  39. 39.

    Robinson, D. A., Monk, A. B., Cooper, J. E., Feil, E. J. & Enright, M. C. Evolutionary genetics of the accessory gene regulator (agr) locus in Staphylococcus aureus. J. Bacteriol. 187, 8312–8321 (2005).

  40. 40.

    Thoendel, M. & Horswill, A. R. Biosynthesis of peptide signals in Gram-positive bacteria. Adv. Appl. Microbiol. 71, 91–112 (2010).

  41. 41.

    Autret, N., Raynaud, C., Dubail, I., Berche, P. & Charbit, A. Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence. Infect. Immun. 71, 4463–4471 (2003).

  42. 42.

    Riedel, C. U. et al. AgrD‐dependent quorum sensing affects biofilm formation, invasion, virulence and global gene expression profiles in Listeria monocytogenes. Mol. Microbiol. 71, 1177–1189 (2009).

  43. 43.

    Vivant, A.-L., Garmyn, D., Gal, L. & Piveteau, P. The Agr communication system provides a benefit to the populations of Listeria monocytogenes in soil. Front. Cell. Infect. Microbiol. 4, 160 (2014).

  44. 44.

    Zetzmann, M., Sánchez-Kopper, A., Waidmann, M. S., Blombach, B. & Riedel, C. U. Identification of the agr peptide of Listeria monocytogenes. Front. Microbiol. 7, 989 (2016).

  45. 45.

    Piewngam, P. et al. Pathogen elimination by probiotic Bacillus via signalling interference. Nature 562, 532–537 (2018).

  46. 46.

    Lee, A. S. et al. Methicillin-resistant Staphylococcus aureus. Nat. Rev. Dis. Primers 4, 18033 (2018).

Download references

Acknowledgements

We thank P. Martín-Gago for fruitful input and T.W. Muir for encouraging comments. P. S. Andersen is acknowledged for providing bacterial strains. This work was supported by the Carlsberg Foundation (2013-01-0333 to C.A.O.) and University of Copenhagen (PhD fellowship to B.H.G.).

Author information

B.H.G. and C.A.O. conceptualized the study. B.H.G., M.S.B., P.P. and M.B. performed the experiments. B.H.G. and C.A.O. wrote the original draft of the manuscript. B.H.G., M.S.B., H.I. and C.A.O. reviewed and edited the final manuscript. C.A.O. acquired funding. H.I. and C.A.O. provided resources and materials. H.I. and C.A.O. supervised the study.

Competing interests

The authors declare no competing interests.

Correspondence to Christian A. Olsen.

Supplementary information

  1. Supplementary Information

    Supplementary experimental data, chemical compound characterization data, Supplementary Figs. 1–26, Supplementary Tables 1–3 and copies of 1H and 13C NMR spectra.

  2. Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1: Overview of the reported workflow.
Fig. 2: NCL trapping and sequence-guided identification of AIP-II (2).
Fig. 3: Synthesis of AIPs.
Fig. 4: Detection limit of NCL trapping for synthetic L. monocytogenes AIP (20).