title: Glycoengineering of host mimicking type-2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces.
abstract: Bacterial carbohydrate structures play a central role in mediating a variety of host-pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram-negative bacteria, is composed of a lipid A-core and the O-antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Galβ1-4(Fucα1-3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram-negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O-antigen. A ubiquitous building block in mammalian N-linked protein glycans is Galβ1-4GlcNAc, referred to as a type-2 N-acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A-cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly-LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI-TOF and NMR analysis. Glycoengineered LOS induced pro-inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.
authors.affiliation: ETH Zurich, Institute of Microbiology, Wolfgang-Pauli-Str. 10, HCI F 406, CH- 8093 Zurich, Switzerland. manuela.mally@glycovaxyn.com
authors.forename: Manuela
authors.initials: M
authors.lastname: Mally
authors.forename: Carolina
authors.initials: C
authors.lastname: Fontana
authors.forename: Salomé
authors.initials: S
authors.lastname: Leibundgut-Landmann
authors.forename: Lamia
authors.initials: L
authors.lastname: Laacisse
authors.forename: Yao-Yun
authors.initials: YY
authors.lastname: Fan
authors.forename: Göran
authors.initials: G
authors.lastname: Widmalm
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D003713:Dendritic Cells / Q000276:immunology / Q000382:microbiology; D004187:Disaccharides / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism; D016480:Helicobacter pylori / Q000378:metabolism; D054884:Host-Pathogen Interactions; D016256:Lewis X Antigen / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D012486:Salmonella typhimurium / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012550:Schistosoma mansoni / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D004187:Disaccharides; C117073:Gal-GlcNAc-Epo; D016256:Lewis X Antigen; D008070:Lipopolysaccharides