Peptidoglycan Fragment Library to Investigate Innate Immune Responses

PROJECT SUMMARY Bacterial cells surround themselves with a peptidoglycan (PG) cell wall, an essential structure that provides cell shape, and resists changes in osmotic pressure and other environmental insults. In addition, PG is highly inflammatory and an important trigger of innate immune responses across the animal kingdom. However, study of these important PG fragments has been hampered by the lack of reproducible, chemically defined, and high quality reagents. We hypothesize that the natural diversity of the generated PG fragments poses unique challenges to the innate immune system that utilizes specific uptake, transport, and receptor systems for sensing and responding to the presence of the bacterial cell wall derived molecules. The goal of this proposal is to develop a biologically relevant PG fragment library and PG synthesis strategies to facilitate a more precise understanding of PG mediated immune responses in the context of health (homeostasis) and inflammatory diseases. In the initial funding period, we used our PG library and genome wide transcriptome analysis to uncover that distinct PG fragments indeed induce specific immune responses, through related but distinct PG sensing receptors. We were successful in our aims to produce a fragment library, print a PG array, share the fragment library with multiple laboratories and to discover that specific immune receptors recognize discrete peptidoglycan fragments. Transporters responsible for the cellular internalization of specific PG fragments were defined and novel innate immune sensing complexes were identified. The project has now developed into a mechanistic phase with a keen focus on intestinal innate immunity and inflammation, where our team of chemical biologists and immunologists will work together to innovate PG production strategies and fine tune PG probes to better understand the specificity of their trafficking and signaling modalities. Ultimately, we aim to learn the molecular requirements for cellular uptake and innate immune recognition of a variety of PG fragments, especially in mucosal macrophages, by the characterizing the SLC transporters involved and the key innate immune receptors - PGLYRPs and the NOD proteins required. This renewal aims to enhance the PG library with additional modalities for partner capture (both in cellular, animal models and array format), synthesize and screen for inhibitors for bacterial cell wall uptake and recognition, and define the pathways driving specific gene expression outputs, all building upon the results from the previous funding cycle: Aim 1: Identification and fermentation of biologically relevant PG fragments, Aim 2: Defining requirements and mechanisms for PG fragments entering macrophages Aim 3. Define the role(s) of mammalian Peptidoglycan Recognition Proteins (PGLYRPs) in innate immunity and mucosal protection. Ultimately, this dynamic team will set the stage for the future development of small molecule modulators, PG based immunotherapies, adjuvants, and biomarkers while at the same time, sharing new tools with the microbial and immunological communities.