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

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 Burkholderia 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 in 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 confirm the production of glycoproteins with similar glycans in the Burkholderia species B. thailandensis, B. gladioli, and B. pseudomallei. Further, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates

Loss ofO-linked protein glycosylation inBurkholderia cenocepaciaimpairs biofilm formation and siderophore production via alteration of quorum sensing regulation

“…Although these results support the breakdown of BCAL1086 as a consequence of the loss of glycosylation, an alternative explanation is that the changes in degradation arise from alterations in protease levels or activities in the ∆pglL mutant. Previously, we reported that ∆pglL results in enhanced casein proteolytic activity (65). However, our global proteome analysis shows only modest changes in protease levels.…”

“…We previously demonstrated that loss of glycosylation causes defects in motility (56), reduction of virulence in plant and insect infection models (56,65), and defects in carbon utilisation (65). To better understand the role of glycosylation in B. cenocepacia we assessed the effect of loss of glycosylation on the proteome.…”

“…To better understand the role of glycosylation in B. cenocepacia we assessed the effect of loss of glycosylation on the proteome. To achieve this we generated markerless deletion mutants in the O-oligosaccharyltransferase pglL (∆pglL; BCAL0960 (56)) gene, the recently identified O-linked glycan cluster (∆OGC; BCAL3114 to BCAL3118) responsible for the generation of the glycan used for O-linked glycosylation (65), and a double glycosylation null strain (∆pglL∆OGC). We also constructed a chromosomal pglL complemented strain (∆pglL amrAB::S7-pglL-his10, Supplementary Figure 1A).…”

“…Previously, we reported B. cenocepacia possesses an O-linked glycosylation system responsible for the modification of at least 23 proteins with a trisaccharide glycan using the enzyme PglL (BCAL0960) (56). Building on this work we recently identified the biosynthetic locus, the O-glycosylation cluster (OGC, BCAL3114 to BCAL3118) responsible for the generation of the O-linked glycan, established the O-linked glycan structure as β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc, and demonstrated that glycosylation was required for optimal bacterial fitness and resistance to clearance in Galleria mellonella infection models (65).…”

Open database searching enables the identification and comparison of bacterial glycoproteomes without defining glycan compositions prior to searching

“…While glycosylation in bacteria was first identified in the 1970s [20], it is only within the last two decades that it has become clear that this class of modifications is ubiquitous across bacterial genera [16, 18, 21]. Unlike Eukaryotic systems, which utilise a relatively small set of monosaccharides, bacterial glycoproteins are decorated with a diverse range of monosaccharides [22] leading to a staggering array of glycan structures [23–32]. This glycan diversity represents a significant challenge to the field as it makes the identification of novel bacterial glycoproteins a non-trivial analytical undertaking.…”