Breaking down Bacterial Cell Walls to Understand Inflammation
Bacterial cell wall biosynthesis ranks among the top targets for antibiotics. The bacterial cell wall, a polymer of carbohydrate and peptides, makes an excellent antibiotic target for two reasons: (1) it is essential for bacteria and (2) humans do not have bacterial cell walls – thus the drugs do not harm human cells. In addition to serving as a target for antibiotics, the human innate immune system uses the bacterial cell wall as a molecular calling card to recognize their presence and subsequently generate the appropriate immune response. We are interested in understanding how the bacterial cell wall is processed both by bacteria and the human host and propose new methods and tools for the characterization of this important polymer. Both commensal and pathogenic bacteria are believed to produce peptidoglycan fragments and misrecognition can lead to the development of inflammatory bowel disease (IBD), such as Crohn's disease (CD), asthma and gastrointestinal (GI) cancers. Importantly, a long-standing debate around the biological relevance of the immunoactive synthetic fragment muramyl dipeptide (MDP) remains unclear due to a lack of NAM-based probes. We hypothesize that there are unidentified enzymatic targets and bacterial cell wall fragments that will be useful in the design of novel antibiotics and anti-inflammatory therapies.
We have taken a two-pronged approach towards testing this hypothesis. From the small molecule side, we have established an in vitro assay, which allows us to assess the affinity of Nod2, an innate immune receptor that binds to bacterial cell wall fragments. This assay has allowed us to tease apart binding from activation and we have begun to derive rules for molecular recognition by intracellular innate immune receptors. In addition, we have developed a robust synthetic method to readily access a library of bacterial cell wall derivatives. These derivatives will be used as affinity reagents to capture both human and bacterial enzymes that are responsible for bacterial cell wall processing. From the larger polymer side, we have embedded carbohydrates with small modifiable tags into the bacterial cell wall. We developed a method to label the NAM glycan backbone of E. coli, P. putida, and B. subtillis in whole cells. The results reveal fundamental architectural details of the glycan chains of the peptidoglycan, and further enable us to track the engulfment and breakdown of bacteria by macrophages, ultimately revealing a peptidoglycan digestion mechanism for invasive bacteria.
Schaefer AK, Melnyk JE, Baksh MM, Lazor KM, Finn MG, Grimes CL. Membrane Association Dictates Ligand Specificity for the Innate Immune Receptor NOD2 ACS Chem Biol. 12, 2216-2224 (2017)
Liang H, DeMeester KE, Hou CW, Parent MA, Caplan JL, Grimes CL. Metabolic labeling of the carbohydrate core in bacterial peptidoglycan and its applications. Nat Commun. 8, 15015 (2017)
Lauro ML, D'Ambrosio EA, Bahnson BJ, Grimes CL. Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2. ACS Infect Dis. 3, 264-270 (2017)
Melnyk, J.E., Mohanan, V., Schaefer, A.K., Hou, C-H., Grimes, C.L. Peptidoglycan Modifications Tune the Stability and Function of the Innate Immune Receptor Nod2", J Am Chem Soc, 137, 6987 (2015)