Glycoproteomic and proteomic analysis of reveals glycosylation events within FliF and MotB are dispensable for motility.

UNLABELLED

Across the Burkholderia genus -linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in complex species, such as , little is known about how specific glycosylation sites impact protein functionality. Within this study, we sought to improve our understanding of the breadth, dynamics, and requirement for glycosylation across the glycoproteome. Assessing the glycoproteome across different culture media using complementary glycoproteomic approaches, we increase the known glycoproteome to 141 glycoproteins. Leveraging this repertoire of glycoproteins, we quantitively assessed the glycoproteome of using Data-Independent Acquisition (DIA) revealing the glycoproteome is largely stable across conditions with most glycoproteins constitutively expressed. Examination of how the absence of glycosylation impacts the glycoproteome reveals that the protein abundance of only five glycoproteins (BCAL1086, BCAL2974, BCAL0525, BCAM0505, and BCAL0127) are altered by the loss of glycosylation. Assessing Δ (ΔBCAL0525), Δ (ΔBCAL0127), and ΔBCAM0505 strains, we demonstrate the loss of FliF, and to a lesser extent MotB, mirror the proteomic effects observed in the absence of glycosylation in Δ. While both MotB and FliF are essential for motility, we find loss of glycosylation sites in MotB or FliF does not impact motility supporting these sites are dispensable for function. Combined this work broadens our understanding of the glycoproteome supporting that the loss of glycoproteins in the absence of glycosylation is not an indicator of the requirement for glycosylation for protein function.

IMPORTANCE

is an opportunistic pathogen of concern within the Cystic Fibrosis community. Despite a greater appreciation of the unique physiology of gained over the last 20 years a complete understanding of the proteome and especially the O-glycoproteome, is lacking. In this study, we utilize systems biology approaches to expand the known glycoproteome as well as track the dynamics of glycoproteins across growth phases, culturing media and in response to the loss of glycosylation. We show that the glycoproteome of is largely stable across conditions and that the loss of glycosylation only impacts five glycoproteins including the motility associated proteins FliF and MotB. Examination of MotB and FliF shows, while these proteins are essential for motility, glycosylation is dispensable. Combined this work supports that glycosylation can be dispensable for protein function and may influence protein properties beyond stability.