Abstract
We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of Gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria.
Highlights
Teichoic acids are anionic carbohydrate-containing polymers present in the cell wall of many Gram-positive bacteria, and encompass both wall teichoic acids (WTAs) as well as lipoteichoic acids (LTAs)
The WTAs are covalently bound to the peptidoglycan by phosphodiester bonds between N-acetylmuramic acid and a special linkage unit, whereas LTAs are amphipathic molecules tethered to the cytoplasmic membrane via a glycolipid moiety [1,2,3,4,5]
Different Listeria strains were chosen for the analysis of their WTA composition
Summary
Teichoic acids are anionic carbohydrate-containing polymers present in the cell wall of many Gram-positive bacteria, and encompass both wall teichoic acids (WTAs) as well as lipoteichoic acids (LTAs). The WTAs are covalently bound to the peptidoglycan by phosphodiester bonds between N-acetylmuramic acid and a special linkage unit, whereas LTAs are amphipathic molecules tethered to the cytoplasmic membrane via a glycolipid moiety [1,2,3,4,5]. Membrane-associated LTAs show less structural and compositional diversity than WTAs [6,7]. Peptidoglycan-associated WTAs are highly variable in structure, and often feature species- or even strain-specific variations. Most frequently, they are comprised of polyglycerol-phosphate (GroP) or polyribitol-phosphate (RboP) chains both of which can be decorated with a variety of different sugars and/or esterified with D-alanine [8,9]. WTAs composed of poly(RboP) are produced by important human pathogens such as Staphylococcus aureus, Staphylococcus saprophyticus, and Listeria monocytogenes [10]
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