Advertisement

Peptidoglycan Isolation and Binding Studies with LysM-Type Pattern Recognition Receptors

  • Ute Bertsche
  • Andrea A. GustEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)

Abstract

In the last decade, more and more plant receptors for complex carbohydrate structures have been described. However, studies on receptor binding to glycan ligands are often hampered due to the technical challenge to obtain pure preparations of homogeneous carbohydrate ligands such as bacterial peptidoglycan (PGN) in amounts suitable for studying protein–glycan interactions. Also, most approaches rely on the availability of defined soluble ligands, which in the case of glycans can rarely be synthesized but have to be purified from the respective microorganism. In this chapter, we describe the purification of complex PGN from sources such as gram-positive bacteria, from which PGN isolation is facilitated due to its larger content in their cell wall. Insoluble PGN can subsequently be used in simple carbohydrate pull-down assays to test for interaction with plant proteins. In this respect, lysin motif (LysM)-domain containing proteins are of particular interest. All plant receptors described to date to be involved in the perception of N-Acetylglucosamine-containing ligands (such as PGN or chitin) have been shown to belong to this protein class. Thus, this chapter will also include the production of recombinant LysM proteins to analyze their PGN interaction.

Key words

Peptidoglycan Chitin LysM Carbohydrate affinity assay Protein–glycan interaction 

Notes

Acknowledgments

We thank the Deutsche Forschungsgemeinschaft (SFB 766) for support to U.B. and A.A.G. Roland Willmann is acknowledged for preparing Fig. 1 and for helpful discussions on the manuscript.

References

  1. 1.
    Turner RD, Vollmer W, Foster SJ (2014) Different walls for rods and balls: the diversity of peptidoglycan. Mol Microbiol 91:862–874CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Vollmer W, Seligman SJ (2010) Architecture of peptidoglycan: more data and more models. Trends Microbiol 18:59–66CrossRefPubMedGoogle Scholar
  3. 3.
    Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477PubMedPubMedCentralGoogle Scholar
  4. 4.
    de Pedro MA, Cava F (2015) Structural constraints and dynamics of bacterial cell wall architecture. Front Microbiol 6:449CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    de Jonge BL, Chang YS, Gage D, Tomasz A (1992) Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A. J Biol Chem 267:11248–11254PubMedGoogle Scholar
  6. 6.
    Glauner B (1988) Separation and quantification of muropeptides with high-performance liquid chromatography. Anal Biochem 172:451–464CrossRefPubMedGoogle Scholar
  7. 7.
    Kühner D, Stahl M, Demircioglu DD, Bertsche U (2014) From cells to muropeptide structures in 24 h: peptidoglycan mapping by UPLC-MS. Sci Rep 4:7494CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bertsche U, Mayer C, Götz F, Gust AA (2015) Peptidoglycan perception--sensing bacteria by their common envelope structure. Int J Med Microbiol 305:217–223CrossRefPubMedGoogle Scholar
  9. 9.
    Dworkin J (2014) The medium is the message: interspecies and interkingdom signaling by peptidoglycan and related bacterial glycans. Annu Rev Microbiol 68:137–154CrossRefPubMedGoogle Scholar
  10. 10.
    Felix G, Boller T (2003) Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco. J Biol Chem 278:6201–6208CrossRefPubMedGoogle Scholar
  11. 11.
    Erbs G, Silipo A, Aslam S, De Castro C, Liparoti V, Flagiello A, Pucci P, Lanzetta R, Parrilli M, Molinaro A, Newman MA, Cooper RM (2008) Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity. Chem Biol 15:438–448CrossRefPubMedGoogle Scholar
  12. 12.
    Gust AA, Biswas R, Lenz HD, Rauhut T, Ranf S, Kemmerling B, Gotz F, Glawischnig E, Lee J, Felix G, Nurnberger T (2007) Bacteria-derived peptidoglycans constitute pathogen-associated molecular patterns triggering innate immunity in Arabidopsis. J Biol Chem 282:32338–32348CrossRefPubMedGoogle Scholar
  13. 13.
    Millet YA, Danna CH, Clay NK, Songnuan W, Simon MD, Werck-Reichhart D, Ausubel FM (2010) Innate immune responses activated in Arabidopsis roots by microbe-associated molecular patterns. Plant Cell 22:973–990CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Liu B, Li JF, Ao Y, Qu J, Li Z, Su J, Zhang Y, Liu J, Feng D, Qi K, He Y, Wang J, Wang HB (2012) Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity. Plant Cell 24:3406–3419CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wheeler R, Chevalier G, Eberl G, Gomperts Boneca I (2014) The biology of bacterial peptidoglycans and their impact on host immunity and physiology. Cell Microbiol 16:1014–1023CrossRefPubMedGoogle Scholar
  16. 16.
    Ao Y, Li Z, Feng D, Xiong F, Liu J, Li JF, Wang M, Wang J, Liu B, Wang HB (2014) OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity. Plant J 80:1072–1084CrossRefPubMedGoogle Scholar
  17. 17.
    Willmann R, Lajunen HM, Erbs G, Newman MA, Kolb D, Tsuda K, Katagiri F, Fliegmann J, Bono JJ, Cullimore JV, Jehle AK, Gotz F, Kulik A, Molinaro A, Lipka V, Gust AA, Nürnberger T (2011) Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. Proc Natl Acad Sci U S A 108:19824–19829CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gust AA, Willmann R, Desaki Y, Grabherr HM, Nürnberger T (2012) Plant LysM proteins: modules mediating symbiosis and immunity. Trends Plant Sci 17:495–502CrossRefPubMedGoogle Scholar
  19. 19.
    Buist G, Steen A, Kok J, Kuipers OR (2008) LysM, a widely distributed protein motif for binding to (peptido)glycans. Mol Microbiol 68:838–847CrossRefPubMedGoogle Scholar
  20. 20.
    Antolin-Llovera M, Petutsching EK, Ried MK, Lipka V, Nurnberger T, Robatzek S, Parniske M (2014) Knowing your friends and foes—plant receptor-like kinases as initiators of symbiosis or defence. New Phytol 204:791–802CrossRefPubMedGoogle Scholar
  21. 21.
    Sanchez-Vallet A, Saleem-Batcha R, Kombrink A, Hansen G, Valkenburg DJ, Thomma BP, Mesters JR (2013) Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. Elife 2:e00790CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Wong JE, Alsarraf HM, Kaspersen JD, Pedersen JS, Stougaard J, Thirup S, Blaise M (2014) Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein. FEBS J 281:1196–1208CrossRefPubMedGoogle Scholar
  23. 23.
    Wong JE, Midtgaard SR, Gysel K, Thygesen MB, Sorensen KK, Jensen KJ, Stougaard J, Thirup S, Blaise M (2015) An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase. Acta Crystallogr D Biol Crystallogr 71:592–605CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    de Jonge R, van Esse HP, Kombrink A, Shinya T, Desaki Y, Bours R, van der Krol S, Shibuya N, Joosten MH, Thomma BP (2010) Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science 329:953–955CrossRefPubMedGoogle Scholar
  25. 25.
    Broghammer A, Krusell L, Blaise M, Sauer J, Sullivan JT, Maolanon N, Vinther M, Lorentzen A, Madsen EB, Jensen KJ, Roepstorff P, Thirup S, Ronson CW, Thygesen MB, Stougaard J (2012) Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding. Proc Natl Acad Sci U S A 109:13859–13864CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Sorensen KK, Simonsen JB, Maolanon NN, Stougaard J, Jensen KJ (2014) Chemically synthesized 58-mer LysM domain binds lipochitin oligosaccharide. Chembiochem (A European journal of chemical biology) 15:2097–2105CrossRefGoogle Scholar
  27. 27.
    Hayafune M, Berisio R, Marchetti R, Silipo A, Kayama M, Desaki Y, Arima S, Squeglia F, Ruggiero A, Tokuyasu K, Molinaro A, Kaku H, Shibuya N (2014) Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization. Proc Natl Acad Sci U S A 111:E404–E413CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Maeda H (1980) A new lysozyme assay based on fluorescence polarization or fluorescence intensity utilizing a fluorescent peptidoglycan substrate. J Biochem 88:1185–1191CrossRefPubMedGoogle Scholar
  29. 29.
    Leppänen A, Cummings RD (2010) Fluorescence-based solid-phase assays to study glycan-binding protein interactions with glycoconjugates. Methods Enzymol 478:241–264CrossRefPubMedGoogle Scholar
  30. 30.
    Maolanon NN, Blaise M, Sorensen KK, Thygesen MB, Clo E, Sullivan JT, Ronson CW, Stougaard J, Blixt O, Jensen KJ (2014) Lipochitin oligosaccharides immobilized through oximes in glycan microarrays bind LysM proteins. Chembiochem (A European journal of chemical biology) 15:425–434CrossRefGoogle Scholar
  31. 31.
    Biswas R, Voggu L, Simon UK, Hentschel P, Thumm G, Götz F (2006) Activity of the major staphylococcal autolysin Atl. FEMS Microbiol Lett 259:260–268CrossRefPubMedGoogle Scholar
  32. 32.
    Petutschnig EK, Jones AM, Serazetdinova L, Lipka U, Lipka V (2010) The lysin motif receptor-like kinase (LysM-RLK) CERK1 is a major chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced phosphorylation. J Biol Chem 285:28902–28911CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Iordanescu S (1975) Host controlled restriction mutants of Staphylococcus aureus. Arch Roum Pathol Exp Microbiol 34:55–58PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Infection Biology, Interfaculty Institute for Microbiology and Infection Medicine Tübingen (IMIT)University of TübingenTübingenGermany
  2. 2.Department of Plant Biochemistry, ZMBPUniversity of TübingenTübingenGermany

Personalised recommendations