Alex is giving his FOE Seminar on Sept 25th

Our very own Alex is defending in approximately two weeks! Here are the details:

Title: Probing for N-acetylglucosamine in the Glycans of Escherichia coli

Date: September 25th, 2023 at 9 am EST

Location: Davenport East Seminar Room, LM Building

Remote Meeting ID: 864 2288 3168 (https://utoronto.zoom.us/j/86422883168) Passcode: 173090

Abstract: The glycobiology of bacteria is fundamental to understanding many aspects of their behaviour and how they interact with their environments, infect hosts, and transition between various lifestyles. Typical lab culturing conditions ignore the often-dominant biofilm lifestyle of bacteria, but it is in that form that many microorganisms survive in nature and persist under the stressful environment of host cells. The altered glycobiology of the sessile cell results in changing glycan distribution levels as extracellular polymeric substances begin to form the nascent biofilm matrix. The important biofilm polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG) was studied using the PNAG hydrolase DspB, developing a PNAG-specific binding probe for specific detection in the heterogeneous setting of a biofilm. Applying this tool to the early stages of biofilm development revealed how PNAG accumulates in areas of locally high concentrations, explaining the effectiveness of DspB binding despite its lower affinity for PNAG oligosaccharides. PNAG is composed of N-acetylglucosamine (GlcNAc), the most ubiquitous member of the amino sugars, which are disproportionately found in many crucial bacterial glycans. The nucleotide sugar substrate for glycan biosynthesis UDP-GlcNAc is a product of the hexosamine biosynthesis pathway and a key target for metabolic glycan labelling. Engineering the hexosamine pathway through introduction of the hexosamine 1-kinase NahK permitted direct feeding of the metabolic analogue N-azidoacetylglucosamine (GlcNAz) and metabolic labelling in E. coli. The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis. In the typical planktonic lifestyle, dissection of the GlcNAc glycome for potential UDP-GlcNAz utilization revealed varying degrees of incorporation in glycan destinations, with the highest levels of incorporation observed in peptidoglycan, and no observable incorporation in the Wzx/Wzy-dependent glycans lipopolysaccharide or enterobacterial common antigen. The lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo corroborated this result. Finally, returning to the biofilm lifestyle, PNAG was found to incorporate low levels of GlcNAz, determined through isolation of labelled biofilm matrix material and through investigations with PNAG synthase in vitro promiscuity towards UDP-GlcNAz. This body of work will guide future developments of carbohydrate-based probes and metabolic engineering strategies in future studies of bacterial glycobiology.