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.
Highlights
Predicted B. cenocepacia O-glycosylation gene cluster is conserved in the Burkholderia genus
We hypothesized that at least four genes encoding a phosphoglycosyltransferase [23, 24], two additional glycosyltransferases, and a flippase protein would be required for the synthesis and translocation of a B. cenocepacia lipid-linked trisaccharide glycan [22, 24]
Chromosome 1 of B. cenocepacia has a putative five-gene operon flanked by the O-antigen synthesis cluster and lipid A-core biosynthesis genes (Fig. 1A) [25], which could be involved in protein glycosylation
Summary
A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans. We report the identification of the Burkholderia O-glycosylation (ogc) cluster, which includes genes in chromosome 1 of B. cenocepacia (BCAL3114 to BCAL3118) encoding the enzymes required for the stepwise assembly of the lipid-linked O-glycan in the cytoplasm and its flipping across the inner membrane. We discovered these genes are present in all species of Burkholderia and confirm biochemically the presence of O-linked proteins in several species of Burkholderia, suggesting protein O-glycosylation is a conserved feature of this genus. We demonstrate that individuals previously infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, and B. mallei develop antiO-glycan serum antibodies, suggesting Burkholderia glycoproteins display a common epitope perceived by the human immune system
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
