Protocol

Bacterial Cell Surfaces

Volume 966 of the series Methods in Molecular Biology pp 289-308

Date:

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

  • Filipe CarvalhoAffiliated withGroup of Molecular Microbiology, Institute for Molecular and Cell Biology
  • , María Graciela PucciarelliAffiliated withDepartamento de Biología Molecular, Universidad Autónoma de MadridCentro Nacional de Biotecnología, Universidad Autónoma de Madrid
  • , Francisco García-del PortilloAffiliated withCentro Nacional de Biotecnología
  • , Didier CabanesAffiliated withGroup of Molecular Microbiology, Institute for Molecular and Cell Biology
  • , Pascale CossartAffiliated withUnité des Interactions Bactéries-Cellules, Institut Pasteur Email author 

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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