Abstract
Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics.
Highlights
Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults
The innate immune system utilizes a series of receptors, including Toll-like receptors and Nod-like receptors[9,10,11], which bind to fragments of PG, such as the synthetic fragments muramyl dipeptide (MDP) and muramyl tripeptide (MTP), to generate the proper immune response[12,13,14] (Fig. 1a)
To answer questions regarding both immune recognition and the three-dimensional (3D) architecture of bacterial cell walls, we developed the synthesis of the necessary PG building blocks, subsequent incorporation strategies and a method to label and visualize the glycan backbone directly
Summary
Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. To answer questions regarding both immune recognition and the three-dimensional (3D) architecture of bacterial cell walls, we developed the synthesis of the necessary PG building blocks, subsequent incorporation strategies and a method to label and visualize the glycan backbone directly. UDP-N-acetyl glucosamine colleagues[37] revealed that the NAG unit of PG could potentially be labelled at the 2-N acetyl position in lactic acid bacteria These elegant methods have proven useful in studying bacterial cell wall. A NAM-based labelling strategy would allow for the selective incorporation of label into NAM residues, which are only found in bacterial PG5
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