Loss ofO-linked protein glycosylation inBurkholderia cenocepaciaimpairs biofilm formation and siderophore production via alteration of quorum sensing regulation
Authors: Oppy Cameron C. , Jebeli Leila , Kuba Miku , Oates Clare V. , Strugnell Richard , Edgington-Mitchell Laura E. , Valvano Miguel A. , Hartland Elizabeth L. , Newton Hayley J. , Scott Nichollas E. 
Published: September 2019

AbstractO-linked protein glycosylation is a conserved feature of theBurkholderiagenus. ForBurkholderia cenocepacia, the addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins is required for virulence and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. This study demonstrates that the globalB. cenocepaciaproteome undergoes dramatic changes consistent with alterations in global transcriptional regulation in the absence of glycosylation. Using luciferase reporter assays and DNA cross-linking analysis, we confirm the repression of the master quorum sensing regulon CepR/I in response to the loss of glycosylation, which leads to the abolition of biofilm formation, defects in siderophore production, and reduced virulence. The abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants except for BCAL1086 and BCAL2974, which were found in reduced amount, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, as well as for reduced virulence and siderophore production. Together, our results show that loss of glycosylation inB. cenocepaciaresults in a global cell reprogramming via alteration of the CepR/I regulon, which cannot be explained by the abundance changes in knownB. cenocepaciaglycoproteins.IMPORTANCEProtein glycosylation is increasingly recognised as a common protein modification in bacterial species. Despite this commonality our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption ofO-linked glycosylation in the opportunistic pathogenBurkholderia cenocepaciausing a combination of proteomic, molecular and phenotypic assays. We find that in contrast to recent findings on theN-linked glycosylation systems ofCampylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that virulence attenuation observed within glycosylation-nullB. cenocepaciastrains are consistent with alteration of the master virulence regulator CepR. The repression of CepR transcription and its associated phenotypes support a model in which the virulence defects observed in glycosylation-null strains are at least in part due to transcriptional alteration and not the direct result of the loss of glycosylationper-se. This research unravels the pleotropic effects ofO-linked glycosylation inB. cenocepacia,demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.