Helicobacter pylori is a multidrug resistant, opportunistic pathogen that causes several gastrointestinal ulcers and cancers. Biomolecules involved in the formation of its glycoproteins are attractive targets for the development of drugs that target H. pylori with high specificity to overcome the damaging effects caused by the use of broad-spectrum antibiotics. Although the glycosylation of H. pylori’s flagellin protein, which facilitates it motility, has been well-studied, its general O-linked protein glycosylation system is still not fully understood. Metabolic Oligosaccharide Engineering (MOE) experiments identified 13 genes that code for distinct glycosyltransferase enzymes involved in H. pylori’s glycosylation pathway, which consists of an overlap between its glycoprotein and lipopolysaccharide synthesis. However, the order in which the enzymes act and the structures of the glycans formed are yet to be elucidated. Bacterial glycans are complex, heterogeneous structures comprised of a more diverse pool of monosaccharide building blocks than eukaryotic glycans. This structural complexity poses a challenge for the use of conventional mass-dependent approaches to studying glycans, which are predominantly tailored to eukaryotic systems. This project utilizes the recently developed, mass-independent approach known as Isotope Targeted Glycoproteomics (IsoTaG) to characterize and compare the glycoproteomes of wildtype H. pylori G27 to two protein glycosylation mutants (Δ579 and Δ580) to begin to gain structural information on the glycans formed and how H. pylori’s general glycosylation pathway proceeds. Initial IsoTaG results have yielded a large number of enriched glycosylated proteins, and we were able to identify glycosylation sites with high confidence in a subset of these hits. Ongoing analysis of glycans at these glycosylation sites across strains will provide incisive insight into H. pylori’s glycosylation system, ultimately unveiling potential targets of glycosylation-based therapeutics.

Applying IsoTaG to understand Helicobacter pylori’s glycoprotein biosynthesis

ACS Spring Meeting 2021

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