Skip to main content
SpringerLink
Log in

Detection of Fibronectin-Binding Proteins of Streptococcus pyogenes Using Ligand Blot Analysis

  • Protocol
  • First Online:
Group A Streptococcus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2136))

Abstract

Streptococcus pyogenes utilizes extracellular cellular matrix (ECM) proteins to adhere to human tissues and internalize into host cells. Fibronectin (Fn) is one of the most abundant ECM proteins and targeted by a wide variety of secreted Fn-binding proteins (Fbps) of S. pyogenes. However, prior to detailed kinetic analysis of that binding process, evaluations of the ability of S. pyogenes strains to bind to Fn as well as interactions of target molecules with Fn are required. In this chapter, we present routine procedures for ligand blot analysis with labeled human Fn, using bacterial cell wall extracts prepared by either enzymatic digestion of cells or extraction with a denaturing agent.

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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

References

  1. Frantz C, Stewart KM, Weaver VM (2010) The extracellular matrix at a glance. J Cell Sci 123:4195–4200. https://doi.org/10.1242/jcs.023820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Schmidt S, Friedl P (2010) Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res 339:83–92. https://doi.org/10.1007/s00441-009-0892-9

    Article  CAS  PubMed  Google Scholar 

  3. Engvall E, Ruoslahti E (1977) Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer 20:1–5. https://doi.org/10.1002/ijc.2910200102

    Article  CAS  PubMed  Google Scholar 

  4. Singh P, Carraher C, Schwarzbauer JE (2010) Assembly of fibronectin extracellular matrix. Annu Rev Cell Dev Biol 26:397–419. https://doi.org/10.1146/annurev-cellbio-100109-104020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Potts JR, Campbell ID (1994) Fibronectin structure and assembly. Curr Opin Cell Biol 6:648–655. https://doi.org/10.1016/0955-0674(94)90090-6

    Article  CAS  PubMed  Google Scholar 

  6. Kuusela P (1978) Fibronectin binds to Staphylococcus aureus. Nature 276:718–720. https://doi.org/10.1038/276718a0

    Article  CAS  PubMed  Google Scholar 

  7. Cue D, Lam H, Cleary PP (2001) Genetic dissection of the Streptococcus pyogenes M1 protein: regions involved in fibronectin binding and intracellular invasion. Microb Pathog 31:231–242. https://doi.org/10.1006/mpat.2001.0467

    Article  CAS  PubMed  Google Scholar 

  8. Pancholi V, Fischetti VA (1992) A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. J Exp Med 176:415–426. https://doi.org/10.1084/jem.176.2.415

    Article  CAS  PubMed  Google Scholar 

  9. Fisher M, Huang YS, Li X et al (2008) Shr is a broad-spectrum surface receptor that contributes to adherence and virulence in group A streptococcus. Infect Immun 76:5006–5015. https://doi.org/10.1128/IAI.00300-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Caswell CC, Oliver-Kozup H, Han R et al (2010) Scl1, the multifunctional adhesin of group A Streptococcus, selectively binds cellular fibronectin and laminin, and mediates pathogen internalization by human cells. FEMS Microbiol Lett 303:61–68. https://doi.org/10.1111/j.1574-6968.2009.01864.x

    Article  CAS  PubMed  Google Scholar 

  11. Hanski E, Caparon M (1992) Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Proc Natl Acad Sci U S A 89:6172–6176. https://doi.org/10.1073/pnas.89.13.6172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Talay SR, Valentin-Weigand P, Jerlstrom PG et al (1992) Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells. Infect Immun 60:3837–3844

    Article  CAS  Google Scholar 

  13. Jaffe J, Natanson-Yaron S, Caparon MG et al (1996) Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains. Mol Microbiol 21:373–384. https://doi.org/10.1046/j.1365-2958.1996.6331356.x

    Article  CAS  PubMed  Google Scholar 

  14. Rocha CL, Fischetti VA (1999) Identification and characterization of a novel fibronectin-binding protein on the surface of group a streptococci. Infect Immun 67:2720–2728

    Article  CAS  Google Scholar 

  15. Terao Y, Kawabata S, Kunitomo E et al (2001) Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator. Mol Microbiol 42:75–86. https://doi.org/10.1046/j.1365-2958.2001.02579.x

    Article  CAS  PubMed  Google Scholar 

  16. Terao Y, Kawabata S, Nakata M et al (2002) Molecular characterization of a novel fibronectin-binding protein of Streptococcus pyogenes strains isolated from toxic shock-like syndrome patients. J Biol Chem 277:47428–47435. https://doi.org/10.1074/jbc.M209133200

    Article  CAS  PubMed  Google Scholar 

  17. Kreikemeyer B, Talay SR, Chhatwal GS (1995) Characterization of a novel fibronectin-binding surface protein in group a streptococci. Mol Microbiol 17:137–145. https://doi.org/10.1111/j.1365-2958.1995.mmi_17010137.x

    Article  CAS  PubMed  Google Scholar 

  18. Rakonjac JV, Robbins JC, Fischetti VA (1995) DNA sequence of the serum opacity factor of group A streptococci: identification of a fibronectin-binding repeat domain. Infect Immun 63:622–631

    Article  CAS  Google Scholar 

  19. Jeng A, Sakota V, Li Z et al (2003) Molecular genetic analysis of a group A Streptococcus operon encoding serum opacity factor and a novel fibronectin-binding protein, SfbX. J Bacteriol 185:1208–1217. https://doi.org/10.1128/JB.185.4.1208-1217.2003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Courtney HS, Li Y, Dale JB et al (1994) Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci. Infect Immun 62:3937–3946

    Article  CAS  Google Scholar 

  21. Mazmanian SK, Liu G, Ton-That H et al (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285:760–763. https://doi.org/10.1126/science.285.5428.760

    Article  CAS  PubMed  Google Scholar 

  22. Schwarz-Linek U, Werner JM, Pickford AR et al (2003) Pathogenic bacteria attach to human fibronectin through a tandem beta-zipper. Nature 423:177–181. https://doi.org/10.1038/nature0189

    Article  CAS  PubMed  Google Scholar 

  23. Molinari G, Rohde M, Guzman CA et al (2000) Two distinct pathways for the invasion of Streptococcus pyogenes in non-phagocytic cells. Cell Microbiol 2:145–154. https://doi.org/10.1046/j.1462-5822.2000.00040.x

    Article  CAS  PubMed  Google Scholar 

  24. Wang B, Yurecko RS, Dedhar S et al (2006) Integrin-linked kinase is an essential link between integrins and uptake of bacterial pathogens by epithelial cells. Cell Microbiol 8:257–266. https://doi.org/10.1111/j.1462-5822.2005.00618.x

    Article  CAS  PubMed  Google Scholar 

  25. Pandiripally V, Gregory E, Cue D (2002) Acquisition of regulators of complement activation by Streptococcus pyogenes serotype M1. Infect Immun 70:6206–6214. https://doi.org/10.1128/IAI.70.11.6206-6214.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Reuter M, Caswell CC, Lukomski S et al (2010) Binding of the human complement regulators CFHR1 and factor H by streptococcal collagen-like protein 1 (Scl1) via their conserved C termini allows control of the complement cascade at multiple levels. J Biol Chem 285:38473–38485. https://doi.org/10.1074/jbc.M110.143727

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hamada S, Horikoshi T, Minami T et al (1989) Purification and characterization of cell-associated glucosyltransferase synthesizing water-insoluble glucan from serotype c Streptococcus mutans. J Gen Microbiol 135:335–344. https://doi.org/10.1099/00221287-135-2-335

    Article  CAS  PubMed  Google Scholar 

  28. Mori Y, Yamaguchi M, Terao Y et al (2012) α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps. J Biol Chem 287:10472–10481. https://doi.org/10.1074/jbc.M111.280321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yokogawa K, Kawata S, Nishimura S et al (1974) Mutanolysin, bacteriolytic agent for cariogenic streptococci: partial purification and properties. Antimicrob Agents Chemother 6:156–165. https://doi.org/10.1128/aac.6.2.156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Courtney HS, Ofek I, Hasty DL (1997) M protein mediated adhesion of M type 24 Streptococcus pyogenes stimulates release of interleukin-6 by HEp-2 tissue culture cells. FEMS Microbiol Lett 151:65–70. https://doi.org/10.1111/j.1574-6968.1997.tb10395.x

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Oral and Molecular Microbiology, Graduate School of Dentistry, Osaka University, Suita-Osaka, Japan

    Masanobu Nakata & Shigetada Kawabata

Authors
  1. Masanobu Nakata
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shigetada Kawabata
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shigetada Kawabata .

Editor information

Editors and Affiliations

  1. School of Medical Sciences, The University of Auckland, Auckland, New Zealand

    Thomas Proft

  2. School of Medical Sciences, The University of Auckland, Auckland, New Zealand

    Jacelyn M. S. Loh

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Nakata, M., Kawabata, S. (2020). Detection of Fibronectin-Binding Proteins of Streptococcus pyogenes Using Ligand Blot Analysis. In: Proft, T., Loh, J. (eds) Group A Streptococcus. Methods in Molecular Biology, vol 2136. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0467-0_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0467-0_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0466-3

  • Online ISBN: 978-1-0716-0467-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation