Catherine Grimes

BREAKING DOWN BACTERIAL CELL WALLS TO UNDERSTAND INFLAMMATION

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. 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.