Loss of O -Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators

Abstract

Protein glycosylation is increasingly recognized as a common posttranslational 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 of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.

A glycoengineered antigen exploiting a conserved proteinO-glycosylation pathway in theBurkholderiagenus for diagnosis of glanders infections

“…The corresponding protein has 3 serine residues that can be O-glycosylated, as demonstrated by mass spectrometry analysis. An optimal O-glycosylation motif, WPAAASAP (the underlined serine is the glycosylation acceptor site), required for recognition by the Neisseria meningitidis PglL O-Tase has been suggested (39), which is very similar to the sequences where we detect glycosylated serine residues in Burkholderia glycoproteins (20,21,32). The presence of three glycosylation sites in BCAL2737a, coupled to its small mass, make this protein an ideal carrier for presenting the trisaccharide.…”

“…2B and D). This was not unexpected since a subset of the Burkholderia glycoproteome is proteolytically degraded (32). Therefore, as a strategy to stabilize BCAL2737a, we also generated versions of this proteins that were N-and C-terminally fused with the cholera toxin B subunit (CtxB) and contained a C-terminal 10x-His tag ( Fig.…”

“…3B, Supplementary Dataset). To further support the degradation of unglycosylated BCAL2737a peptidome datasets from Oppy et al (32) were reanalyzed against a K56-2 proteome containing the BCAL2737a protein sequence. The new analysis identified multiple peptide breakdown products previously not assigned to known proteins, which were more abundant and intense in the glycosylation null strain ΔpglL compared to K56-2 and the pglL complemented strain (Fig.…”

“…proteome containing the BCAL2737a protein sequence, resulting in the identification of multiple peptide breakdown products previously not assigned to known proteins, which are clearly both more numerous and more intense in the glycosylation null strain ΔpglL compared to K56-2 and the pglL complemented strain(32). B, Biological replicate.…”

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses

“…Using ZIC-HILIC enrichment, we have previously shown over 100 proteins are glycosylated within Burkholderia species [44]. Despite this insight, the complete repertoire of most Burkholderia glycoproteomes are still unknown, hampering our understanding of the impacts observed when glycosylation is disrupted [31,45,46]. Within members of the Burkholderia genus, two linear trisaccharides composed of β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc and Suc-β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc (where Suc is Succinyl) are utilized for protein glycosylation [31,35].…”