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

Abstract

The <i>Burkholderia</i> genus encompasses many Gram-negative bacteria living in the rhizosphere. Some <i>Burkholderia</i> species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. <i>Burkholderia cenocepacia O</i>-linked protein glycosylation has been reported, but the chemical structure of the <i>O</i>-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 <i>O</i>-glycans, and NMR-based structural analyses, we identified a <i>B. cenocepacia O</i>-glycosylation (<i>ogc</i>) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked <i>O</i>-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 <i>ogc</i> cluster is conserved in the <i>Burkholderia</i> genus, and we confirm the production of glycoproteins with similar glycans in the <i>Burkholderia</i> species: <i>B. thailandensis</i>, <i>B. gladioli</i>, and <i>B. pseudomallei</i> Furthermore, we show that absence of protein <i>O-</i>glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a <i>Galleria mellonella</i> larva infection model. Finally, our experiments revealed that patients infected with <i>B. cenocepacia</i>, <i>Burkholderia multivorans</i>, <i>B. pseudomallei</i>, or <i>Burkholderia mallei</i> develop <i>O-</i>glycan-specific antibodies. Together, these results highlight the importance of general protein <i>O-</i>glycosylation in the biology of the <i>Burkholderia</i> genus and its potential as a target for inhibition or immunotherapy approaches to control <i>Burkholderia</i> infections.