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Synthesis of bacterial disaccharides and chemical probes for innate immune sensing
Date
2024
Authors
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Publisher
University of Delaware
Abstract
The human innate immune system is the body’s first line of defense against invading pathogens, responding to and differentiating between pathogenic bacteria and the 39 trillion commensal bacteria that compose the human microbiome. Misrecognition of commensal bacteria is implicated in a variety of illnesses such as inflammatory bowel diseases (IBDs), rheumatoid arthritis, and gastrointestinal (GI) cancers. Peptidoglycan (PG), an important element of the bacterial cell wall, is responsible for this differentiation by the immune system. The study of PG is often limited, hampered by the lack of accessible tools; as such, scientists are left to use the commercially available, minimal PG fragment, muramyl dipeptide (MDP). While it is long known that MDP activates a human nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-dependent NFkB innate immune response, this small, synthetic fragment fails to capture the vast diversity of bacterial PG fragments that is sensed by the immune system. ☐ In 2021, we hypothesized that discreet fragments give distinct molecular signatures. Indeed, we demonstrated that disaccharide N-acetylglucosamine N-acetylmuramic acid (GMTP) activates innate immune pathways differently and more robustly compared to MDP, validating that PG signaling is more complex than previously understood. In this thesis, we aim to expand the PG fragment library toolbox which we use to uncover innate immune receptors and untangle downstream signaling pathways. ☐ In an effort to gain better understanding of complex PG sensing by the immune system, we developed: 1) synthetic routes for natural and modified peptidoglycan derivatives (specifically focusing on synthetically complicated disaccharide fragments) which were used to interrogate immune responses, and 2) the development of a novel PG glycan microarray to survey diverse PG chemical space and assess binding specificities. This thesis discusses the application of synthetic peptidoglycan probes, immunological assays and the development of novel tools to describe the interaction between peptidoglycan fragments and innate immune receptors. ☐ The first report of peptidoglycan recognition protein 1 (PGLYRP-1) as an intracellular receptor for disaccharide fragments like GMTP was established in this thesis. We characterized the binding of PGLYRP-1 and GMTP using the microarray platform described in this text. Further we conducted detailed immunological experiments were conducted to elucidate the cellular events. ☐ Modular synthetic routes to complex PG fragments have been developed and are described herein. PG composition varies between bacterial species, further complicating the immune signaling patterns. The identity of fragments, the mechanism by which these molecules are generated, as well as how and when they interact with cellular receptors both in an innate and adaptive responses remains elusive. In this thesis, new synthetic methodologies to access a larger PG fragments were developed. These fragments were then assayed in bone marrow-derived macrophages (BMDMs) to accurately survey the transcriptional response. Upon rigorously testing and optimization, the resultant PG fragment library was subsequently printed on glycan arrays, and binding data was found. The tools developed and experiments performed in this thesis will aid in developing a better understanding how bacteria in the microbiome communicate by demonstrating how bacteria differential shed PG fragments to generate a specific and tuned immune response. These studies provide new insights into our understanding of differential PG innate immune signaling and which is critical to develop novel therapeutics to control inflammation and adjuvants to bolster vaccine response.
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Keywords
Carbohydrate synthesis, Glycan microarray, Peptidoglycan, Bone marrow, Immune system