title: The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering.
abstract: Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Jon
authors.identifier: 0000-0002-4538-4007
authors.initials: J
authors.lastname: Cuccui
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Vanessa S
authors.identifier: 0000-0002-2734-4036
authors.initials: VS
authors.lastname: Terra
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St. Mary's Campus, London W2 1PG, UK.
authors.forename: Janine T
authors.initials: JT
authors.lastname: Bossé
authors.affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
authors.forename: Andreas
authors.identifier: 0000-0002-5938-9124
authors.initials: A
authors.lastname: Naegeli
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Sherif
authors.initials: S
authors.lastname: Abouelhadid
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St. Mary's Campus, London W2 1PG, UK.
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Li
authors.affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
authors.forename: Chia-Wei
authors.initials: CW
authors.lastname: Lin
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Prerna
authors.initials: P
authors.lastname: Vohra
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tucker
authors.affiliation: The Royal Veterinary College, Hawkshead Campus, Hatfield, Hertfordshire AL9 7TA, UK.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Rycroft
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St. Mary's Campus, London W2 1PG, UK.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Langford
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK brendan.wren@lshtm.ac.uk.
authors.forename: Brendan W
authors.identifier: 0000-0002-6140-9489
authors.initials: BW
authors.lastname: Wren
mesh_terms: D000072283:A549 Cells; D016977:Actinobacillus pleuropneumoniae / Q000235:genetics / Q000472:pathogenicity*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006031:Glycosylation; D006801:Humans; D009876:Operon; D015202:Protein Engineering; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors
keywords: Actinobacillus pleuropneumoniae; N-linked glycosylation; adhesion