# Glycoproteomics and Its Role in Understanding Bacterial O‐Linked Glycosylation

## TL;DR

This review explains how O-linked glycosylation in bacteria is studied using glycoproteomics, focusing on enzymes and techniques involved in this process.

## Contribution

The paper provides a comprehensive overview of bacterial O-linked glycosylation systems and glycoproteomic methods, emphasizing recent advancements.

## Key findings

- O-linked glycosylation in bacteria involves enzymes like O-OTases and affects surface or periplasmic proteins.
- Glycoproteomic techniques and bioinformatic tools now enable high-throughput analysis of bacterial O-glycoproteomes.
- The review highlights glycosylation systems in Gram-negative genera such as Burkholderia, Neisseria, and Acinetobacter.

## Abstract

Protein glycosylation is now recognized as a ubiquitous process observed in all domains of life. Within bacterial species, carbohydrates can be attached to multiple residues with glycosylation of serine, threonine, or tyrosine residues via their hydroxyl side chains referred to as O‐linked glycosylation. To date, multiple bacterial enzymes have been identified that mediate O‐linked glycosylation targeting either surface or periplasmic bacterial proteins, and in the case of toxin/effectors, host proteins. Within this review, we discuss the current understanding of common bacterial O‐linked glycosylation systems and the glycoproteomic approaches which have been used to characterize these events. Focusing on O‐oligosaccharyltransferases (O‐OTases), flagellin‐specific glycosylation systems, and glycosyltransferase toxin/effectors, we discuss the characteristics of known glycosylation systems. For the general O‐linked systems mediated by the PglL oligosaccharyltransferases, we outline the key considerations when assessing glycosylation events across the Burkholderia, Neisseria, and Acinetobacter genera. In addition, we highlight the technologies and advancements in glycoproteomic techniques, as well as the bioinformatic tools that now facilitate high throughput studies of O‐linked glycosylation within bacterial species. Combined, this review outlines our current understanding of O‐linked glycosylation within well characterized Gram‐negative genera and the current technologies available for exploring bacterial O‐glycoproteomes.