Multiscale Chemical Biology and Biomolecular Engineering Tools for Studying Bacteria-Immune Cell Interactions

Abstract

Bacterial cells surround themselves with a peptidoglycan (PG) cell wall, an essential structure that resists changes in osmotic pressure and other environmental insults. In the innate immune system, macrophage cells sense fragments of PG to initiate an immune response. The goal of this dissertation is to utilize newly developed PG substrates to remodel and label a wide variety of bacterial species to study their breakdown in macrophages through newly established assays for visualizing these interactions in three-dimensional (3D) culture. We hypothesize that PG diversity, naturally present across the multitude of bacterial species, is essential for generating the correct immune response for host defense. Currently, a major gap in the knowledge surrounds these critical PG fragments such as: the identity of naturally produced immunostimulatory fragments during an immune response, how they are generated, and how they interact with innate immune receptors is not well understood. In this dissertation, a library of robust PG probes was utilized to remodel and visualize the bacterial cell wall in a variety of organisms ranging from Gram-positive and Gram-negative organisms as well as pathogenic and commensal. PG labeling strategies were also expanded to include “click” chemistries that allowed for live cell imaging of bacteria and bacterial invasion. 3D assays were established to culture macrophages in microenvironments inspired by properties of the intestinal tissue in healthy and diseased states. These 2D assays and 3D assays were utilized as invasion models for bacteria to isolate and identify naturally produced bacterial fragments inside of macrophage cells. These bacterial fragments are being analyzed for immunostimulatory activity and compared across species and modifications to the invasion assay to better understand mechanisms related to inflammatory bowel disorders and aid in the development of therapies and antibiotics for misrecognition of these fragments.