Bacterial cells surround themselves with a peptidoglycan (PG) cell wall, an essential structure that resists changes in osmotic pressure and other environmental insults. To a certain degree, PG is also essential to humans as antibiotics target its destruction and fragments activate immune responses. The basic building blocks of PG have been known for over fifty years; however, the higher architectural features of this polymer and complete set of immunostimulating fragments remain unknown. We hypothesize that differences in overall PG structure and fragment generation are important for sensing pathogenic bacteria. The glycan of the PG is essential for immune recognition; study of this important structure has been hampered by a lack of tools to label and track the fate of PG carbohydrate precursors and the resultant polymer in living cells. Currently, researchers are limited to few carbohydrate probes and even fewer larger fragments. Chemical synthesis is laborious and challenging to even expert carbohydrate chemists. Here we will present methodology to label the glycan of the PG in a range of microbes to facilitate identification, tracking, manipulation and analysis of the glycans derived from PG with their biological binding partners and determine their functions. We have utilized a metabolic labeling approach in which the necessary functionalized PG biosynthetic building blocks are synthesized, provided to the microbe and incorporated in the backbone of the polymer. This has not been done before as the synthesis of the UDP-sugar building blocks is challenging and the uptake and processing pathways for the free sugars are not widely distributed. To overcome this challenge we will present a genetic engineering approach using The bacterial PG recycling enzymes, AmgK and MurU. Tagged lactol substrates were provided to cells (Escherichia coli, Bacillus subtilis and the pathogens: Helicobacter pylori (Hp), Staphylococcus aureus and Borrelia burgdorferi, whose genomes have been engineered to encode for AmgK and MurU. Here we aim to showcase the utility of this method for immunologists and microbiologists to use in their studies. This innovative carbohydrate metabolic labeling method for peptidoglycan will be an approachable and powerful technique for researchers.

Metabolic carbohydrate cell wall probes for bacterial structure and immune recognition studies

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.