Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

Nature Chemical Biology volume 14, pages 609–617 (2018)Cite this article

Abstract

Serine hydrolases play diverse roles in regulating host–pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here we describe a chemical proteomic screen that identified ten previously uncharacterized S. aureus serine hydrolases that mostly lack human homologs. We termed these enzymes fluorophosphonate-binding hydrolases (FphA–J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of fphB confirmed that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduced infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Identification of serine hydrolases and inhibitors in live S. aureus by competitive ABPP.
Fig. 2: LC/LC-MS/MS-based identification of serine hydrolases in S. aureus.
Fig. 3: Biochemical characterization of FphB and development of an FphB-selective fluorescent ABP.
Fig. 4: Stimulatory activity of eukaryotic cell components on FphB activity.
Fig. 5: Imaging of FphB-activity using the fluorescent ABP JCP251-bT.
Fig. 6: Effects of loss of FphB activity on infectivity in vivo.

Similar content being viewed by others

References

  1. Reddy, P. N., Srirama, K. & Dirisala, V. R. An update on clinical burden, diagnostic tools, and therapeutic options of Staphylococcus aureus. Infect. Dis. (Auckl.) 10, 1179916117703999 (2017).

    Google Scholar 

  2. Tong, S. Y., Davis, J. S., Eichenberger, E., Holland, T. L. & Fowler, V. G. Jr. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin. Microbiol. Rev. 28, 603–661 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Laupland, K. B. et al. The changing epidemiology of Staphylococcus aureus bloodstream infection: a multinational population-based surveillance study. Clin. Microbiol. Infect. 19, 465–471 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Duthie, E. S. & Lorenz, L. L. Staphylococcal coagulase; mode of action and antigenicity. J. Gen. Microbiol. 6, 95–107 (1952).

    CAS  PubMed  Google Scholar 

  5. Frees, D., Gerth, U. & Ingmer, H. Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus. Int. J. Med. Microbiol. 304, 142–149 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Staub, I. & Sieber, S. A. Beta-lactam probes as selective chemical-proteomic tools for the identification and functional characterization of resistance associated enzymes in MRSA. J. Am. Chem. Soc. 131, 6271–6276 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Frankel, M. B., Hendrickx, A. P., Missiakas, D. M. & Schneewind, O. LytN, a murein hydrolase in the cross-wall compartment of Staphylococcus aureus, is involved in proper bacterial growth and envelope assembly. J. Biol. Chem. 286, 32593–32605 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bukowski, M., Wladyka, B. & Dubin, G. Exfoliative toxins of Staphylococcus aureus. Toxins (Basel) 2, 1148–1165 (2010).

    Article  CAS  Google Scholar 

  9. Pietrocola, G., Nobile, G., Rindi, S. & Speziale, P. Staphylococcus aureus manipulates innate immunity through own and host-expressed proteases. Front. Cell. Infect. Microbiol. 7, 166 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Böttcher, T. & Sieber, S. A. Beta-lactones as specific inhibitors of ClpP attenuate the production of extracellular virulence factors of Staphylococcus aureus. J. Am. Chem. Soc. 130, 14400–14401 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Liu, Y., Patricelli, M. P. & Cravatt, B. F. Activity-based protein profiling: the serine hydrolases. Proc. Natl Acad. Sci. USA 96, 14694–14699 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Patricelli, M. P., Giang, D. K., Stamp, L. M. & Burbaum, J. J. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics 1, 1067–1071 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Simon, G. M. & Cravatt, B. F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J. Biol. Chem. 285, 11051–11055 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Henness, S. & Perry, C. M. Orlistat: a review of its use in the management of obesity. Drugs 66, 1625–1656 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Thornberry, N. A. & Weber, A. E. Discovery of JANUVIA (Sitagliptin), a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Curr. Top. Med. Chem. 7, 557–568 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Kluge, A. F. & Petter, R. C. Acylating drugs: redesigning natural covalent inhibitors. Curr. Opin. Chem. Biol. 14, 421–427 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Adam, G. C., Sorensen, E. J. & Cravatt, B. F. Chemical strategies for functional proteomics. Mol. Cell. Proteomics 1, 781–790 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Leung, D., Hardouin, C., Boger, D. L. & Cravatt, B. F. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 21, 687–691 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Jessani, N., Liu, Y., Humphrey, M. & Cravatt, B. F. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl Acad. Sci. USA 99, 10335–10340 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ortega, C. et al. Systematic survey of serine hydrolase activity in Mycobacterium tuberculosis defines changes associated with persistence. Cell Chem. Biol. 23, 290–298 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tallman, K. R., Levine, S. R. & Beatty, K. E. Small-molecule probes reveal esterases with persistent activity in dormant and reactivating Mycobacterium tuberculosis. ACS Infect. Dis. 2, 936–944 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hatzios, S. K. et al. Chemoproteomic profiling of host and pathogen enzymes active in cholera. Nat. Chem. Biol. 12, 268–274 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zweerink, S. et al. Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea. Nat. Commun. 8, 15352 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hall, C. I. et al. Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion. Proc. Natl Acad. Sci. USA 108, 10568–10573 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Lentz, C. S. et al. Design of selective substrates and activity-based probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect. Dis. 2, 807–815 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cadieux, B., Vijayakumaran, V., Bernards, M. A., McGavin, M. J. & Heinrichs, D. E. Role of lipase from community-associated methicillin-resistant Staphylococcus aureus strain USA300 in hydrolyzing triglycerides into growth-inhibitory free fatty acids. J. Bacteriol. 196, 4044–4056 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rosenstein, R. & Gotz, F. Staphylococcal lipases: biochemical and molecular characterization. Biochimie 82, 1005–1014 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Nguyen, M. T. et al. Staphylococcal (phospho)lipases promote biofilm formation and host cell invasion. Int. J. Med. Microbiol. https://doi.org/10.1016/j.ijmm.2017.11.013 (2017).

  29. Staub, I. & Sieber, S. A. Beta-lactams as selective chemical probes for the in vivo labeling of bacterial enzymes involved in cell wall biosynthesis, antibiotic resistance, and virulence. J. Am. Chem. Soc. 130, 13400–13409 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Fey, P. D. et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4, e00537–e12 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Utaida, S. et al. Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon. Microbiology 149, 2719–2732 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Pietiäinen, M. et al. Transcriptome analysis of the responses of Staphylococcus aureus to antimicrobial peptides and characterization of the roles of vraDE and vraSR in antimicrobial resistance. BMC Genomics 10, 429 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sass, P. et al. The lantibiotic mersacidin is a strong inducer of the cell wall stress response of Staphylococcus aureus. BMC Microbiol. 8, 186 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Muthaiyan, A. et al. Antimicrobial effect and mode of action of terpeneless cold-pressed Valencia orange essential oil on methicillin-resistant Staphylococcus aureus. J. Appl. Microbiol. 112, 1020–1033 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Palazzolo-Ballance, A. M. et al. Neutrophil microbicides induce a pathogen survival response in community-associated methicillin-resistant Staphylococcus aureus. J. Immunol. 180, 500–509 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Bore, E., Langsrud, S., Langsrud, O., Rode, T. M. & Holck, A. Acid-shock responses in Staphylococcus aureus investigated by global gene expression analysis. Microbiology 153, 2289–2303 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Neumann, Y. et al. The effect of skin fatty acids on Staphylococcus aureus. Arch. Microbiol. 197, 245–267 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Garland, M. et al. Development of an activity-based probe for acyl-protein thioesterases. PLoS One 13, e0190255 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Jinno, A. & Park, P. W. Role of glycosaminoglycans in infectious disease. Methods Mol. Biol. 1229, 567–585 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. van Gemst, J. J. et al. RNA contaminates glycosaminoglycans extracted from cells and tissues. PLoS One 11, e0167336 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Raz, A. & Fischetti, V. A. Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane. Proc. Natl Acad. Sci. USA 105, 18549–18554 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Mazmanian, S. K., Liu, G., Jensen, E. R., Lenoy, E. & Schneewind, O. Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc. Natl Acad. Sci. USA 97, 5510–5515 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Clarke, A. J. & Dupont, C. O-Acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can. J. Microbiol. 38, 85–91 (1992).

    Article  CAS  PubMed  Google Scholar 

  44. Bera, A., Biswas, R., Herbert, S. & Gotz, F. The presence of peptidoglycan O-acetyltransferase in various staphylococcal species correlates with lysozyme resistance and pathogenicity. Infect. Immun. 74, 4598–4604 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Brown, S. et al. Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids. Proc. Natl Acad. Sci. USA 109, 18909–18914 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Winstel, V. et al. Wall teichoic acid glycosylation governs Staphylococcus aureus nasal colonization. MBio 6, e00632–15 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Weadge, J. T. & Clarke, A. J. Identification and characterization of O-acetylpeptidoglycan esterase: a novel enzyme discovered in Neisseria gonorrhoeae. Biochemistry 45, 839–851 (2006).

    Article  CAS  PubMed  Google Scholar 

  48. Weadge, J. T., Pfeffer, J. M. & Clarke, A. J. Identification of a new family of enzymes with potential O-acetylpeptidoglycan esterase activity in both Gram-positive and Gram-negative bacteria. BMC Microbiol. 5, 49 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Rahman, M. M. et al. The Staphylococcus aureus methicillin resistance factor FmtA is a d-amino esterase that acts on teichoic acids. MBio 7, e02070–e15 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Garcia-Fernandez, E. et al. Membrane microdomain disassembly inhibits MRSA antibiotic resistance. Cell 171, 1354–1367.e20 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Pang, Y. Y. et al. agr-dependent interactions of Staphylococcus aureus USA300 with human polymorphonuclear neutrophils. J. Innate Immun. 2, 546–559 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Weerapana, E., Speers, A. E. & Cravatt, B. J. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)-a general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2, 1414–1425 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Horswill (University of Iowa) for sharing the GFP plasmid pCM29. We thank N. Amara and J. Yim for help with NMR analyses and S. Chen for LC-MS analysis of JCP678, and L. Popov, O. Zurek, J. Romaniuk and L. Cegelski for discussions. We also thank E. Yeh for access to the BD Accuri flow cytometer. C.S.L. was supported through a postdoctoral research fellowship by the German Research Foundation (DFG). This work was further supported through NIH grants 1R01GM111703 and R01EB026332 to M.B., 1R01GM117004 and 1R01GM118431-01A1 to E.W., 1R01AI101171 and 1R01AI069233 to E.P.S., and R21AI117255 to M.R.A.

Author information

Author notes

  1. These authors contributed equally: Christian S. Lentz, Jessica R. Sheldon.

Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Christian S. Lentz, Megan Garland & Matthew Bogyo

  2. Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

    Jessica R. Sheldon & Eric P. Skaar

  3. Department of Chemistry, Boston College, Chestnut Hill, MA, USA

    Lisa A. Crawford & Eranthie Weerapana

  4. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Rachel Cooper & Manuel R. Amieva

  5. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Manuel R. Amieva & Matthew Bogyo

Authors
  1. Christian S. Lentz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jessica R. Sheldon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lisa A. Crawford
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rachel Cooper
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Megan Garland
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Manuel R. Amieva
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eranthie Weerapana
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Eric P. Skaar
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Matthew Bogyo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.S.L. and M.B. conceived the project. C.S.L designed and performed the in vitro experiments, synthesized compounds and analyzed data. J.R.S. designed and performed the in vivo infection experiments and the genetic manipulation of S. aureus, and analyzed data. L.A.C. and E.W. performed LC-MS/MS analysis. R.C. contributed to the comparative bacterial labeling experiments. M.G. synthesized compounds. M.R.A. contributed to the experimental design and analyzed data. E.P.S. designed and analyzed in vivo infection experiments. M.B. supervised the project, designed experiments and analyzed data. C.S.L. and M.B. wrote the manuscript, and all authors reviewed, discussed and edited the manuscript.

Corresponding author

Correspondence to Matthew Bogyo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lentz, C.S., Sheldon, J.R., Crawford, L.A. et al. Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP). Nat Chem Biol 14, 609–617 (2018). https://doi.org/10.1038/s41589-018-0060-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0060-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing