Characterization and application of peptidoglycan O-acetyltransferase B utilizing N-acetylcysteamine derivatives
Date
2017
Authors
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Publisher
University of Delaware
Abstract
Bacteria have the natural ability to install protective post-synthetic modifications onto its bacterial peptidoglycan (PG); the coat that is woven into bacterial cell wall. Peptidoglycan O-acetyltransferase B (PatB) catalyzes the O-acetylation of peptidoglycan in Gram (–) bacteria, which aids in bacterial survival, as it prevents autolysins such as lysozyme from cleaving the peptidoglycan at the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). In this dissertation, the substrate promiscuity and mechanistic details of PatB’s acetylation function was explored and it was determined that PatB has substrate tolerance for bioorthgonal short N-acetylcysteamine (SNAc) donors. Exploiting this lax specificity, a variety of functionality including azides and alkynes were installed on tri-N-acetylglucosamine (NAG)3, a peptidoglycan mimic, as well as peptidoglycan isolated from various Gram (+) (Bacillus subtilis) and Gram (–) (Escherichia coli, Vibrio parahaemolyticus, and Pseudomonas putida) bacterial species. The bioorthogonal modifications were shown to protect the isolated peptidoglycan against lysozyme degradation in vitro. We further demonstrate that this post-synthetic modification of peptidoglycan can be extended to use click chemistry to fluorescently label the carbohydrate backbone of mature peptidoglycan in whole bacterial cells of Bacillus subtilis. Modifying peptidoglycan post-synthetically can aid in the development of antibiotics and immune modulators by expanding on the current understanding of how the bacterial peptidoglycan is processed by lytic enzymes presented in the innate immune system.
Description
Keywords
Pure sciences, Bacteria labeling, N-acetylcysteamine, O-acetylation, PatB, Peptidoglycan