title: Selective biochemical labeling of Campylobacter jejuni cell-surface glycoconjugates.
abstract: The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni. Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention; however, our understanding of the roles that glycosylation plays in bacteria still pales in comparison with our understanding of mammalian glycosylation. One of the reasons for this is that carbohydrate metabolic labeling, a powerful tool for studying mammalian glycans, is difficult to establish in bacterial systems and has a significantly more limited scope. Herein, we report the development of an alternative strategy that can be used to study bacterial cell-surface glycoconjugates. Galactose oxidase (GalO) is used to generate an aldehyde at C-6 of terminal GalNAc residues of C. jejuni glycans. This newly generated aldehyde can be conjugated with aminooxy-functionalized purification tags or fluorophores. The label can be targeted towards specific glycoconjugates using C. jejuni mutant strains with N-glycan or lipo-oligosaccharides (LOS) assembly defects. GalO-catalyzed labeling of cell-surface glycoproteins with biotin, allowed for the purification and identification of known extracellular N-linked glycoproteins as well as a recently identified O-linked glycan modifying PorA. To expand the scope of the GalO reaction, live-cell fluorescent labeling of C. jejuni was used to compare the levels of surface-exposed LOS to the levels of N-glycosylated, cell-surface proteins. While this study focuses on the GalO-catalyzed labeling of C. jejuni, it can in principle be used to evaluate glycosylation patterns and identify glycoproteins of interest in any bacteria.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Garrett E
authors.initials: GE
authors.lastname: Whitworth
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA imper@mit.edu.
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
mesh_terms: D001710:Biotin / Q000378:metabolism; D016123:Campylobacter jejuni / Q000378:metabolism* / Q000502:physiology; D002240:Carbohydrate Sequence; D006001:Glycoconjugates / Q000378:metabolism*; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data
chemical_list: D006001:Glycoconjugates; D001710:Biotin
keywords: bacterial glycoconjugates; galactose oxidase; glycoprotein identification; lipo-oligosaccharides; live-cell labeling