Poster Presentation 31st Annual Lorne Proteomics Symposium 2026

Understanding Glycosylation Regulation: Impact of Flippase Modulations on Mediating O-glycan Toxicity Within Burkholderia cenocepacia (#15)

Tak To Duncan Ho 1 , Leila Jebeli 1 , Nichollas E. Scott 1
  1. Department of Microbiology and Immunology, The University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

Burkholderia cenocepacia possesses a highly conserved O-linked protein glycosylation pathway essential for virulence and bacterial fitness 1,2. Within this system the five gene cluster known as the O-Glycosylation Cluster (OGC, ogcX,A,B,E,I) facilitates the synthesis of a β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc trisaccharide utilised for protein glycosylation 1. Previous studies in B. cenocepacia have reported conflicting results regarding the essentiality of two genes within the OGC, the putative glycosyltransferase OgcA thought to be required for the attachment of the final carbohydrate in the O-linked glycan, and the putative flippase OgcX suspected to be required for O-linked glycan translocation 1. Within glycan pathways conditional essentiality can occur when blockages during glycan biosynthesis are unable to be resolved. This leads to non-cognate or truncated glycans which cannot be translocated resulting in the accumulation of dead-end glycan intermediates 3,4. As dead-end intermediates sequester the undecaprenyl (Und-P) lipid carrier pool this drives cellular viability, morphology and growth defects 3,4. Within this work we assess the role of OgcX in glycan mediated conditional essentiality within  B. cenocepacia and how OgcX controls the specificity of glycan translocation. We demonstrate that ogcA or ogcX are essential when glycosylation is initiated and the loss of either component is deleterious to B. cenocepacia. We further reveal complementation or overexpression of OgcX alleviates defects due to loss of ogcX/ogcA. Proteomic analysis reveals overexpression of the OgcX flippase results in a relaxed glycan specificity allowing translocation of incomplete or truncated glycans. Finally, we show that modulating glycan specificity alleviates the membrane permeability defects in mutants lacking ogcA, resulting in improved tolerance against membrane stressors. These findings support the sequestration of the Und-P pool with incomplete O-linked glycans is responsible for membrane defects if the OGC biosynthesis pathway is compromised and that the OgcX flippase functions as a quality control mechanism for the O-linked glycosylation system in the Burkholderia genus.

  1. [1] Fathy MY, Scott NE, Molinaro A, et al. (2019) “A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans”, Journal of Biological Chemistry, 294(36):13248–13268.
  2. [2] Oppy CC, Jebeli L, Kuba M, et al. (2019) “Loss of O-Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators”, mSphere, 4(6): e00660-19.
  3. [3] Qin J, Hong Y, Totsika M. (2024) “Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.” Applied and Environmental Microbiology.90(6):e02203-23.
  4. [4] Liu MA, Stent TL, Hong Y and Reeves PR (2015) “Inefficient translocation of a truncated O unit by a Salmonella Wzx affects both O-antigen production and cell growth”, FEMS Microbiology Letters, 362(9).