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mSphere. 2019 Nov 13;4(6). pii: e00660-19. doi: 10.1128/mSphere.00660-19.

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

Author information

1
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia.
2
Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.
3
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
4
Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, Bluestone Center for Clinical Research, New York, New York, USA.
5
Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom.
6
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
7
Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia.
8
Department of Molecular and Translational Science, Monash University, Clayton, Victoria, Australia.
9
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia Nichollas.scott@unimelb.edu.au.
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Contributed equally

Abstract

O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, 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 amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins.IMPORTANCE 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.

KEYWORDS:

Burkholderia cenocepacia ; CepR; DNA binding; glycoproteins; glycosylation; pathogenesis; posttranslational modifications; protein modification; proteomics

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