Extraction of Cell Wall-Bound Teichoic Acids and Surface Proteins from Listeria monocytogenes
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 wordsPeptidoglycan Wall teichoic acids Surface proteins Covalent anchorage Digestion Alkaline hydrolysis PAGE Alcian blue Silver staining
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.
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