Extraction of Cell Wall-Bound Teichoic Acids and Surface Proteins from Listeria monocytogenes

  • Filipe Carvalho
  • María Graciela Pucciarelli
  • Francisco García-del Portillo
  • Didier Cabanes
  • Pascale Cossart
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 966)

Abstract

Gram-positive bacteria contain a cell wall consisting of a thick peptidoglycan layer decorated with surface proteins and polysaccharide-based polymers. The latter include the wall teichoic acids (WTAs), which are anionic glycopolymers covalently linked to the peptidoglycan matrix. They are constituted by a long backbone containing sugars of various sizes (trioses to hexoses) which can be reduced (polyols as in Listeria) or oxidized (uronic acids) and can undergo a variety of species- or often strain-specific modifications and substitutions. These confer unique biochemical properties to WTAs and any defect in the modification or substitution process can potentially affect their biological role in the overall cell wall physiology. Surface proteins can be associated to the cell wall by covalent bonds that anchor the protein to the peptidoglycan lattice. Due to the chemical nature of this bond, covalently bound proteins “co-purify” with peptidoglycan sacculi and are intrinsically insoluble at high temperatures and/or in the presence of ionic detergents. Analysis of this type of proteins therefore requires enzymatic digestion of peptidoglycan for the subsequent release of associated proteins. In contrast, proteins associated to the cell wall by non-covalent interactions are easier to isolate using ionic detergents. In this chapter, we describe methods for the extraction and analysis of (i) WTAs, (ii) covalently, and (iii) non-covalently cell wall-bound surface proteins from the Gram-positive pathogen Listeria monocytogenes.

Key words

Peptidoglycan Wall teichoic acids Surface proteins Covalent anchorage Digestion Alkaline hydrolysis PAGE Alcian blue Silver staining 

Notes

Acknowledgment

This work was supported by grants from the FCT (PTDC/SAU-MIC/111581/2009FCOMP-01-0124-FEDER-015844, PTDC/BIA-BCM/111215/2009FCOMP-01-0124-FEDER-014178, PTDC/BIA-BCM/100088/2008FCOMP-01-0124-FEDER-008860, and ERANet Pathogenomics Listress ERA-PTG/0003/2010 to F.C. and D.C., and PhD fellowship SFRH/BD/61825/2009 to F.C.), from the Spanish Ministry of Economy and Competitiveness (BIO2010-18962 to M.G.P. and PIM2010EPA-00714 to F.G.P.), and from Institut Pasteur, Inserm, INRA ERC (Advanced Grant 233343) and Fondation les Mousquetaires to P.C.

References

  1. 1.
    Pucciarelli MG, Bierne H, García-del Portillo F (2007) The cell wall of Listeria monocytogenes and its role in pathogenicity. In: Goldfine H, Shen H (eds) Listeria monocytogenes: pathogenesis and host response. Springer, New York, pp 81–110. doi: DOI:10.1007/978-0-387-49376-3_5 CrossRefGoogle Scholar
  2. 2.
    Weidenmaier C, Peschel A (2008) Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions. Nat Rev Microbiol 6(4):276–287. doi: 10.1038/nrmicro1861 PubMedCrossRefGoogle Scholar
  3. 3.
    Camejo A, Carvalho F, Reis O, Leitao E, Sousa S, Cabanes D (2011) The arsenal of virulence factors deployed by Listeria monocytogenes to promote its cell infection cycle. Virulence 2(5):379–394. doi: 10.4161/viru.2.5.17703 PubMedCrossRefGoogle Scholar
  4. 4.
    Meredith TC, Swoboda JG, Walker S (2008) Late-stage polyribitol phosphate wall teichoic acid biosynthesis in Staphylococcus aureus. J Bacteriol 190(8):3046–3056. doi: 10.1128/JB.01880-07 PubMedCrossRefGoogle Scholar
  5. 5.
    Endl J, Seidl HP, Fiedler F, Schleifer KH (1983) Chemical composition and structure of cell wall teichoic acids of staphylococci. Arch Microbiol 135(3):215–223PubMedCrossRefGoogle Scholar
  6. 6.
    Pollack JH, Ntamere AS, Neuhaus FC (1992) D-alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin. J Gen Microbiol 138(5):849–859PubMedCrossRefGoogle Scholar
  7. 7.
    Pollack JH, Neuhaus FC (1994) Changes in wall teichoic acid during the rod-sphere transition of Bacillus subtilis 168. J Bacteriol 176(23):7252–7259PubMedGoogle Scholar
  8. 8.
    Min H, Cowman MK (1986) Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: application to electrophoretic analysis of molecular weight distribution. Anal Biochem 155(2):275–285PubMedCrossRefGoogle Scholar
  9. 9.
    Wolters PJ, Hildebrandt KM, Dickie JP, Anderson JS (1990) Polymer length of teichuronic acid released from cell walls of Micrococcus luteus. J Bacteriol 172(9):5154–5159PubMedGoogle Scholar
  10. 10.
    Marraffini LA, Dedent AC, Schneewind O (2006) Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 70(1):192–221. doi: 10.1128/MMBR.70.1.192-221.2006 PubMedCrossRefGoogle Scholar
  11. 11.
    Calvo E, Pucciarelli MG, Bierne H, Cossart P, Albar JP, Garcia-Del Portillo F (2005) Analysis of the Listeria cell wall proteome by two-dimensional nanoliquid chromatography coupled to mass spectrometry. Proteomics 5(2):433–443. doi: 10.1002/pmic.200400936 PubMedCrossRefGoogle Scholar
  12. 12.
    Pucciarelli MG, Calvo E, Sabet C, Bierne H, Cossart P, Garcia-del Portillo F (2005) Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis. Proteomics 5(18):4808–4817. doi: 10.1002/pmic.200402075 PubMedCrossRefGoogle Scholar
  13. 13.
    Garcia-del Portillo F, Calvo E, D’Orazio V, Pucciarelli MG (2011) Association of ActA to peptidoglycan revealed by cell wall proteomics of intracellular Listeria monocytogenes. J Biol Chem 286(40):34675–34689. doi: 10.1074/jbc.M111.230441 PubMedCrossRefGoogle Scholar
  14. 14.
    Scott JR, Barnett TC (2006) Surface proteins of gram-positive bacteria and how they get there. Annu Rev Microbiol 60:397–423. doi: 10.1146/annurev.micro.60.080805.142256 PubMedCrossRefGoogle Scholar
  15. 15.
    Bierne H, Cossart P (2007) Listeria monocytogenes surface proteins: from genome predictions to function. Microbiol Mol Biol Rev 71(2):377–397. doi: 10.1128/MMBR.00039-06 PubMedCrossRefGoogle Scholar
  16. 16.
    Cabanes D, Dussurget O, Dehoux P, Cossart P (2004) Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence. Mol Microbiol 51(6):1601–1614PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Filipe Carvalho
    • 1
  • María Graciela Pucciarelli
    • 2
    • 3
  • Francisco García-del Portillo
    • 4
  • Didier Cabanes
    • 1
  • Pascale Cossart
    • 5
  1. 1.Group of Molecular MicrobiologyInstitute for Molecular and Cell BiologyPortoPortugal
  2. 2.Departamento de Biología MolecularUniversidad Autónoma de MadridMadridSpain
  3. 3.Centro Nacional de BiotecnologíaUniversidad Autónoma de MadridMadridSpain
  4. 4.Centro Nacional de BiotecnologíaMadridSpain
  5. 5.Unité des Interactions Bactéries-CellulesInstitut PasteurParisFrance

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