Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni

Cain, Joel A.; Dale, Ashleigh L.; Niewold, Paula; Klare, William P.; Man, Lok; White, Melanie Y.; Scott, Nichollas E.; Cordwell, Stuart J.

doi:10.1074/mcp.ra118.001199

Cited by 71 publications

(90 citation statements)

References 69 publications

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“…We provide evidence that loss of protein glycosylation causes global metabolic defects in B. cenocepacia concerning the utilization of several different carbon sources and tolerance to osmotic and oxidative stress. Therefore, general protein O-glycosylation is required for metabolic fitness, possibly because it is involved in the stability of its protein targets, as shown in C. jejuni (46,47). We also found that not only are glycosylation defective mutants less virulent in the Galleria mellonella infection model, but also that the pathogen-host interaction at the cellular level is different.…”

Section: Discussionmentioning

confidence: 52%

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

Mohamed

Scott

Molinaro

et al. 2019

Journal of Biological Chemistry

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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

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Section: Discussionmentioning

confidence: 52%

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

Mohamed

Scott

Molinaro

et al. 2019

Journal of Biological Chemistry

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“…This glycan diversity represents a significant challenge to the field as it makes the identification of novel bacterial glycoproteins a non-trivial analytical undertaking. Yet, through advancements in mass spectrometry (MS) [28, 30, 33, 34], these once obscure modifications are increasingly recognisable and are now known to be essential for bacterial fitness [26, 35–38]. Despite our ability to generate rich bacterial glycopeptide data the field still largely uses manual interrogation to identify and characterise novel glycosylation systems [23–32].…”

Section: Introductionmentioning

confidence: 99%

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

Izaham

Scott

2020

Preprint

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Mass spectrometry has become an indispensable tool for the characterisation of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. While glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses prior to database searching. Utilising this approach, we demonstrate how wide tolerance searching can be used to compare glycan utilisation across bacterial species by examining the glycoproteomes of eight Burkholderia species (B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.

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“…Only recently have mechanistic insights emerged on how the loss of glycosylation affects bacterial physiology and pathogenesis. In Campylobacter jejuni, the loss of glycosylation results in decreased stability of the majority of known glycoproteins, which in turn affects virulence (63,64). These data support a model whereby bacterial N-linked glycosylation contributes to protein stability, but it is unclear whether other glycosylation systems, such as O-linked glycosylation, have evolved to stabilize glycosylated proteins.…”

Section: Introductionmentioning

confidence: 81%

“…Acinetobacter baumannii (55) and C. jejuni (63), but the link of this phenotype to alterations in regulations have not previously documented. Previous studies in B. cenocepacia have identified that not all CepR/I regulated proteins are required for biofilm formation.…”

Section: The Observation That Biofilm Formation Is Reduced In Pgll Mmentioning

confidence: 99%

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

Oppy

Jebeli

Kuba

et al. 2019

Preprint

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O-linked protein glycosylation is a conserved feature of the Burkholderia genus. For Burkholderia 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 global B. cenocepacia proteome 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 resultsshow that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the CepR/I regulon, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins. 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 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 virulence attenuation observed within glycosylation-null B. cenocepacia strains 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 glycosylation per-se. This research unravels the pleotropic 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.

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Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni

Cited by 71 publications

References 69 publications

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

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

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

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

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