Fluorescent Labeling of the Carbohydrate Backbone of Peptidoglycan to Track Degradation In Vivo

Microbe associated molecular patterns (MAMPs) are detected via Toll‐ and Nod‐like receptors. Upon their activation, inflammatory molecules are produced in cells. One specific example of a MAMP is muramyl dipeptide (MDP), a small fragment of the bacterial cell wall peptidoglycan. Synthetic MDP has been shown to elicit an immune response by activating nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2). However it is still unclear how MDP, with its unusual stereochemistry, is released from the peptidoglycan. Furthermore, it is unknown if there are other peptidoglycan fragments that can activate the innate immune system. The main objective of this study is to investigate the possibility of using peptidoglycan O ‐acetyltransferase B (PatB) to fluorescently label the peptidoglycan to track the generation of these immunostimulatory fragments. PatB acetylates the sixth hydroxyl position of N ‐acetylmuramic acid (MurNAc), one of the two alternating sugar units of the peptidoglycan backbone, to block the cleavage of the peptidoglycan by lysozyme and other lytic transglycosylases. We aim to hijack this modification pathway to fluorescently label the peptidoglycan. To this end, we optimized the expression and purification of PatB. Initial in vitro studies using PatB and tri‐ N ‐acetylglucosamine (GlcNAc) 3 , the other sugar unit, as the acceptor shows that we are able to install a variety of functional groups, including an alkyl derivative, at the sixth hydroxyl group. The alkyl handle will allow for fluorescent labeling of the peptidoglycan through Click chemistry. We are currently working towards observing the degradation of fluorescently labeled peptidoglycan in vivo . Funding for this project has been provided by NIH COBRE grants and the NIH‐CBI T32 Training Grant.