Bacterial Cell Wall Remodeling to Study the Production of Immunoactivating Fragments

Abstract

The human innate immune system is the front line of defense to protect the body from bacterial infection. One mechanism of activation involves various immunoactivating fragments derived from bacterial cell wall engaging the appropriate receptor to trigger an inflammatory response. Muramyl dipeptide (MDP), a small fragment of bacterial cell wall has been shown to have immunoactivating properties via the innate immune receptor Nod2. Deficiency in sensing MDP may cause serious inflammatory bowel disease (IBD) such as Crohn's Disease (CD). However, how MDP and other immunostimulatory fragments are generated is not well understood. The discovery of new immunoactivating fragments is critical to understand the interactions between human innate immune system and the bacteria it encounters (commensal or pathogenic). Here a combination of organic synthesis, biochemistry and genetic engineering approaches were implemented to bioorthogonally label bacterial peptidoglycan. We have synthesized a variety of azide functionalized cell wall intermediates, including: D‐form amino acids and UDP‐sugar substrates. The peptidoglycan biosynthetic enzymes were purified and assayed for substrate promiscuity. As these enzymes showed relaxed substrate specificity, bacterial cells were grown in the presence of the azide containing precursors. The cells were then labeled with a bioorthogonal azide‐alkyne click reaction to link a fluorescent molecule onto the cell wall. The bacterial cell walls were successfully visualized. Using this method, we will further study the interaction of human innate immune cells and fluorophore labeled bacterial cells to reveal new immunoactivating molecules.Support or Funding InformationFunding of this project is provided by Centers of Biomedical Research Excellence (COBRE)