Skip to main content
SpringerLink
Log in

Activity-Based Protein Profiling in Bacteria

  • Protocol
  • First Online:
Activity-Based Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1491))

Abstract

Understanding the molecular mechanisms of bacterial pathogenesis and virulence is of great importance from both an academic and clinical perspective, especially in view of an alarming increase in bacterial resistance to existing antibiotics and antibacterial agents. Use of small molecules to dissect the basis of these dynamic processes is a very attractive approach due to their ability for rapid spatiotemporal control of specific biochemical functions. Activity-based protein profiling (ABPP), employing small molecule probes to interrogate enzyme activities in complex proteomes, has emerged as a powerful tool to study bacterial pathogenesis. In this chapter, we present a set of ABPP methods to identify and analyze enzymes essential for growth, metabolism and virulence of different pathogens including S. aureus and L. monocytogenes using natural product-inspired activity-based probes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

ABPP:

Activity-based protein profiling

BCA:

Bicinchoninic acid

CC:

Click chemistry

ClpP :

Caseinolytic protein protease P

DMSO:

Dimethyl sufoxide

DTT:

1,4-dithio-d-threitol

HMP:

4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate

Hyd:

Hydrolase CocE/NonD family

KAS II:

β-Ketoacyl acyl carrier protein synthase II

KAS II:

I β-Ketoacyl acyl carrier protein synthase III

Lip:

Lipase

LLO:

Listeriolysin L

LPL:

Lysophospholipase

MRSA :

Methicillin resistant Staphylococcus aureus

MS:

Mass spectrometry

Mur1/2:

UDP-N-Acetylglucosamine 1-carboxyvinyltransferases ½

PBP:

Penicillin-binding protein

PBS :

Phosphate buffered saline

PI-PLC :

Phosphatidylinositol-specific phospholipase C

PL:

Pyridoxal

SDS -PAGE :

Sodium dodecyl polyacrylamide electrophoresis

TAMRA:

5(6)-carboxytetramethylrhodamine

TBE:

Tributyrin esterase

TBTA :

Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine

TCEP :

Tris(2-carboxyethyl)phosphine

TF:

Trigger factor

References

  1. Chubukov V, Gerosa L, Kochanowski K, Sauer U (2014) Coordination of microbial metabolism. Nat Rev Microbiol 12(5):327–340. doi:10.1038/nrmicro3238

    Article  CAS  PubMed  Google Scholar 

  2. Typas A, Banzhaf M, Gross CA, Vollmer W (2012) From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol 10(2):123–136. doi:10.1038/nrmicro2677

    CAS  Google Scholar 

  3. Frees D, Qazi SN, Hill PJ, Ingmer H (2003) Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence. Mol Microbiol 48(6):1565–1578

    Article  CAS  PubMed  Google Scholar 

  4. Brotz-Oesterhelt H, Sass P (2010) Postgenomic strategies in antibacterial drug discovery. Future Microbiol 5(10):1553–1579. doi:10.2217/fmb.10.119

    Article  PubMed  Google Scholar 

  5. Rasko DA, Sperandio V (2010) Anti-virulence strategies to combat bacteria-mediated disease. Nat Rev Drug Discov 9(2):117–128. doi:10.1038/nrd3013

    Article  CAS  PubMed  Google Scholar 

  6. Evans MJ, Cravatt BF (2006) Mechanism-based profiling of enzyme families. Chem Rev 106(8):3279–3301. doi:10.1021/cr050288g

    Article  CAS  PubMed  Google Scholar 

  7. Nodwell MB, Sieber SA (2012) ABPP methodology: introduction and overview. Top Curr Chem 324:1–41. doi:10.1007/128_2011_302

    Article  CAS  PubMed  Google Scholar 

  8. Puri AW, Bogyo M (2013) Applications of small molecule probes in dissecting mechanisms of bacterial virulence and host responses. Biochemistry 52(35):5985–5996. doi:10.1021/bi400854d

    Article  CAS  PubMed  Google Scholar 

  9. Bottcher T, Sieber SA (2008) Beta-lactones as privileged structures for the active-site labeling of versatile bacterial enzyme classes. Angew Chem Int Ed Engl 47(24):4600–4603. doi:10.1002/anie.200705768

    Article  PubMed  Google Scholar 

  10. Bottcher T, Sieber SA (2008) Beta-lactones as specific inhibitors of ClpP attenuate the production of extracellular virulence factors of Staphylococcus aureus. J Am Chem Soc 130(44):14400–14401. doi:10.1021/ja8051365

    Article  PubMed  Google Scholar 

  11. Bottcher T, Sieber SA (2009) Structurally refined beta-lactones as potent inhibitors of devastating bacterial virulence factors. Chembiochem 10(4):663–666. doi:10.1002/cbic.200800743

    Article  PubMed  Google Scholar 

  12. Zeiler E, Korotkov VS, Lorenz-Baath K, Bottcher T, Sieber SA (2012) Development and characterization of improved beta-lactone-based anti-virulence drugs targeting ClpP. Bioorg Med Chem 20(2):583–591. doi:10.1016/j.bmc.2011.07.047

    Article  CAS  PubMed  Google Scholar 

  13. Bottcher T, Sieber SA (2009) Beta-lactones decrease the intracellular virulence of Listeria monocytogenes in macrophages. ChemMedChem 4(8):1260–1263. doi:10.1002/cmdc.200900157

    Article  PubMed  Google Scholar 

  14. Zeiler E, Braun N, Bottcher T, Kastenmuller A, Weinkauf S, Sieber SA (2011) Vibralactone as a tool to study the activity and structure of the ClpP1P2 complex from Listeria monocytogenes. Angew Chem Int Ed Engl 50(46):11001–11004. doi:10.1002/anie.201104391

    Article  CAS  PubMed  Google Scholar 

  15. Zeiler E, List A, Alte F, Gersch M, Wachtel R, Poreba M, Drag M, Groll M, Sieber SA (2013) Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad. Proc Natl Acad Sci U S A 110(28):11302–11307. doi:10.1073/pnas.1219125110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kunzmann MH, Staub I, Bottcher T, Sieber SA (2011) Protein reactivity of natural product-derived gamma-butyrolactones. Biochemistry 50(5):910–916. doi:10.1021/bi101858g

    Article  CAS  PubMed  Google Scholar 

  17. Kunzmann MH, Sieber SA (2012) Target analysis of alpha-alkylidene-gamma-butyrolactones in uropathogenic E. coli. Mol Biosyst 8(11):3061–3067. doi:10.1039/c2mb25313e

    Article  CAS  PubMed  Google Scholar 

  18. Kunzmann MH, Bach NC, Bauer B, Sieber SA (2014) alpha-Methylene-gamma-butyrolactones attenuate Staphylococcus aureus virulence by inhibition of transcriptional regulation. Chem Sci 5(3):1158–1167. doi:10.1039/c3sc52228h

    Article  CAS  Google Scholar 

  19. Staub I, Sieber SA (2008) 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(40):13400–13409. doi:10.1021/ja803349j

    Article  CAS  PubMed  Google Scholar 

  20. Staub I, Sieber SA (2009) 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(17):6271–6276. doi:10.1021/ja901304n

    Article  CAS  PubMed  Google Scholar 

  21. Kolb R, Bach NC, Sieber SA (2014) beta-Sultams exhibit discrete binding preferences for diverse bacterial enzymes with nucleophilic residues. Chem Commun 50(4):427–429. doi:10.1039/c3cc46002a

    Article  CAS  Google Scholar 

  22. Bottcher T, Sieber SA (2010) Showdomycin as a versatile chemical tool for the detection of pathogenesis-associated enzymes in bacteria. J Am Chem Soc 132(20):6964–6972. doi:10.1021/ja909150y

    Article  PubMed  Google Scholar 

  23. Nodwell MB, Menz H, Kirsch SF, Sieber SA (2012) Rugulactone and its analogues exert antibacterial effects through multiple mechanisms including inhibition of thiamine biosynthesis. Chembiochem 13(10):1439–1446. doi:10.1002/cbic.201200265

    Article  CAS  PubMed  Google Scholar 

  24. Nodwell MB, Koch MF, Alte F, Schneider S, Sieber SA (2014) A subfamily of bacterial ribokinases utilizes a hemithioacetal for pyridoxal phosphate salvage. J Am Chem Soc 136(13):4992–4999. doi:10.1021/ja411785r

    Article  CAS  PubMed  Google Scholar 

  25. Battenberg OA, Yang YL, Verhelst SHL, Sieber SA (2013) Target profiling of 4-hydroxyderricin in S. aureus reveals seryl-tRNA synthetase binding and inhibition by covalent modification. Mol Biosyst 9(3):343–351. doi:10.1039/c2mb25446h

    Article  CAS  PubMed  Google Scholar 

  26. Pitscheider M, Mausbacher N, Sieber SA (2012) Antibiotic activity and target discovery of three-membered natural product-derived heterocycles in pathogenic bacteria. Chem Sci 3(6):2035–2041. doi:10.1039/c2sc20290e

    Article  CAS  Google Scholar 

  27. Orth R, Bottcher T, Sieber SA (2010) The biological targets of acivicin inspired 3-chloro- and 3-bromodihydroisoxazole scaffolds. Chem Commun 46(44):8475–8477. doi:10.1039/c0cc02825h

    Article  CAS  Google Scholar 

  28. Eirich J, Orth R, Sieber SA (2011) Unraveling the protein targets of vancomycin in living S. aureus and E. faecalis cells. J Am Chem Soc 133(31):12144–12153. doi:10.1021/ja2039979

    Article  CAS  PubMed  Google Scholar 

  29. Breinbauer R, Vetter IR, Waldmann H (2002) From protein domains to drug candidates – Natural products as guiding principles in the design and synthesis of compound libraries. Angew Chem Int Ed 41(16):2879–2890

    Article  Google Scholar 

  30. Bottcher T, Pitscheider M, Sieber SA (2010) Natural products and their biological targets: proteomic and metabolomic labeling strategies. Angew Chem Int Ed 49(15):2680–2698. doi:10.1002/anie.200905352

    Article  Google Scholar 

  31. Brown DG, Lister T, May-Dracka TL (2014) New natural products as new leads for antibacterial drug discovery. Bioorg Med Chem Lett 24(2):413–418. doi:10.1016/j.bmcl.2013.12.059

    Article  CAS  PubMed  Google Scholar 

  32. Krysiak J, Breinbauer R (2012) Activity-based protein profiling for natural product target discovery. Activity-Based Protein Profiling. Top Curr Chem 324:43–84. doi:10.1007/128_2011_289

Download references

Acknowledgements

We gratefully acknowledge funding from the Deutsche Forschungsgemeinschaft through SFB1035, SFB749, FOR1406, CIPSM, and from the European Research Council (ERC starting grant to S.A.S.). We thank Dr. Megan H. Wright for insightful advices and careful reading of the manuscript.

Author information

Authors and Affiliations

  1. Chair of Organic Chemistry II, TU München, Lichtenbergstraße 4, D-85747, Garching, Germany

    Joanna Krysiak Ph.D. & Stephan A. Sieber Ph.D.

Authors
  1. Joanna Krysiak Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephan A. Sieber Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stephan A. Sieber Ph.D. .

Editor information

Editors and Affiliations

  1. Bio-Organic Synthesis Group Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

    Herman S. Overkleeft

  2. Bio-Organic Synthesis Group Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

    Bogdan I. Florea

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Krysiak, J., Sieber, S.A. (2017). Activity-Based Protein Profiling in Bacteria. In: Overkleeft, H., Florea, B. (eds) Activity-Based Proteomics. Methods in Molecular Biology, vol 1491. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6439-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6439-0_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6437-6

  • Online ISBN: 978-1-4939-6439-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation