In Gram (–) bacteria, peptidoglycan O-acetyltransferase B (PatB) is responsible for the O-acetylation of peptidoglycan to prevent degradation by lysozyme and other lytic transglycosylases generated by its host. In this work, PatB was exploited as a tool to incorporate bioorthogonal functionality on the carbohydrate backbone of the peptidoglycan by using a variety of donors, either p-nitrophenyl (pNP) or N-acetylcysteamine (SNAc). Using tri-N-acetylglucosamine (GlcNAc)3, as a peptidoglycan mimic, a variety of functionalities were installed, including an alkyne and azide handle, on this mimic. Further studies in vitro showed that these modifications have a protective effect on isolated peptidoglycan from B. subtilis, E. coli, P. putida, and V. parahaemolyticus against lysozyme degradation. Intrigued by these results, an alkyne handle was subsequently installed on whole bacterial cells of B. subtilis using the SNAc alkyne derivative and the carbohydrate backbone was fluorescently labeled via click chemistry. Peptidoglycan based fragments, such as muramyl dipeptide (MDP), are known to activate a Nucleotide-binding oligomerization domain-containing protein 2 (Nod2) immune response. Using PatB as a tool, future studies include isolating larger peptidoglycan fragments in order to expand our synthetic library, which includes a variety of muramyl dipeptide (MDP) and N-acetylglucosamine muramyl dipeptide (GMDP) derivatives. These larger fragments will be tested for their ability to initiate an immune response, as well as applied to our peptidoglycan microarray to probe binding with Nod2 and other innate immune receptors.

Use of bioorthogonal N-acetylcysteamine (SNAc) analogues and peptidoglycan O-acetyltransferase B (PatB) to label bacterial peptidoglycan

256th National Meeting (2018)

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