Synthesis and Development of peptidoglycan fragment microarray and probes to investigate innate immune signaling

Abstract

The innate immune system's interaction with bacteria plays a pivotal role in a variety of human diseases, such as Crohn's disease, asthma, and rheumatoid arthritis. In order to develop better therapies, small molecule probes derived from bacterial cell wall peptidoglycan (PG) are needed. Consequently, we are synthesizing new chemical probes based on PG to better understand their interactions with innate immune receptors. The objective here is to develop novel techniques for the production of cell wall derived libraries and then use those molecules as tools for the investigation of innate immune signaling. It has long been known that a small piece of the bacterial cell wall, muramyl‐dipeptide (MDP), has immune stimulatory properties. More specifically, MDP has been found to bind to Nucleotide Oligomerization Domain‐containing protein 2 (NOD2) to initiate a Nod2‐dependent immune response. The power of microarrays lays in their unprecedented capacity to simultaneously interrogate tens to hundreds of thousands of immobilized probes. Here we developed a synthesis for amine functionalized PG derivatives with varying linker length and position, and applied them to NHS‐functionalized glass slides to investigate their interactions with Nod2, and other nod‐like proteins. N‐acetyl muramic acid (NAM) serves as a key building blocks PG fragments. We report a synthetic strategy to access biorthogonal 3‐lactic acid NAM derivatives in large quantities. By utilizing metabolic cell wall recycling and biosynthetic machineries reported previously in our lab, the NAM derivatives were installed into the backbone of Gram‐positive and Gram‐negative bacteria. The whole cells are labelled via click chemistry and visualized using super‐resolution microscopy.Support or Funding InformationCobre (Center of Biomedical Research Excellence at the University of Delaware);NSF (National Science Foundation);U01(NIH) grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.