Glycoproteomic and Proteomic analysis of Burkholderia cenocepacia reveals glycosylation events within FliF and MotB are dispensable for motility. - DIA analysis of glycoprotein mutants

ABSTRACT: Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Burkholderia Cenocepacia

SUBMITTER: Nichollas Scott  

LAB HEAD: Nichollas Scott

PROVIDER: PXD043197 | Pride | 2024-04-17

REPOSITORIES: Pride