A general protein O- glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans

J Biol Chem. 2019 Sep 6;294(36):13248-13268. doi: 10.1074/jbc.RA119.009671. Epub 2019 Jul 26.

Abstract

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

Keywords: Burkholderia; bacteria; cystic fibrosis; galleria mellonella; glanders; glycosylation; immunogenicity; melioidosis; nuclear magnetic resonance (NMR); phenotypic arrays.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Burkholderia / metabolism*
  • Chromatography, Liquid
  • Computational Biology
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Glycosylation
  • Humans
  • Mass Spectrometry
  • Mutation
  • Polysaccharides / analysis
  • Polysaccharides / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Species Specificity

Substances

  • Bacterial Proteins
  • Glycoproteins
  • Polysaccharides
  • Recombinant Proteins