Peptidoglycan fragment microarray platform for human immune system investigation

Abstract

The human immune system's interaction with the bacterial cell wall plays a pivotal role in a variety of human diseases, such as Lyme Arthritis and Crohn's disease. The component of the bacterial cell wall, peptidoglycan (PG), is a mesh-like polymer made up of repeat carbohydrate units crosslinked by peptide chains. However, there are modifications on the carbohydrate and atypical peptides among pathogenic and commensal strains of bacteria. The study of this critical structure has been hampered by a lack of chemical probes derived from PG and tools for high throughput monitoring of PG interactions in both innate and adaptive immunity. Consequently, new chemical probes were synthesized based on PG to study their response against serum antibodies and immune proteins. The array takes advantage of the ability to produce synthetic PG fragments with amine linkage points (Chapter 3). The microarray assay was validated using a monoclonal antibody (mAb), 2E7, that targets muramyl dipeptide (MDP), a canonical minimal immunostimulatory structure of PG. The data showed that the immune system generates different pools of IgG versus IgA among individuals, suggesting that these pools could serve as a potential diagnostic tool for personal therapy (Chapter 2). ☐ To better assess the immunostimulatory properties of these fragments, we sought to develop the synthesis of the fragments, which would be released from the pathogenic bacteria. One crucial fragment is a product release from the lytic transglycosylases, 1,6-anhydro-disaccharide fragments; this feature is present in Tacheal Cytotoxin (TCT). The synthesis route of this important immunological fragment is 20-30 steps in good to excellent yields at a multi-gram scale. Moreover, the synthesis offers several derivation points presenting the opportunity for future probe development and medicinal chemistry campaigns (Chapter 5). ☐ To better understand the natural PG fragments released from the bacterial cell wall, our research laboratory developed a methodology to install bioorthogonal handles into the carbohydrate backbone of the PG. The probe with an azido group labeled on the 3-OH group of the MurNAc expanded the biological toolbox from the initial report of 2-amino probes. This MurNAc derivative was installed into the backbone of the bacterial cell wall and verified using mass spectrometry. Whole cells were fluorescently labeled via click chemistry and visualized using super-resolution microscopy. This bioorthogonal labeling strategy will help to address fundamental questions surrounding PG's role in immunology and microbiology, such as the physiology-relevant immunostimulatory fragments and tracking of bacteria PG in macrophage model systems (Chapter 4). ☐ The synthetic PG fragment library and the microarray incorporated with those fragments provide a platform to investigate human innate and adaptive immunity against bacterial muropeptides. Ultimately, this dissertation reveals "antibody fingerprints" and gene induction diversities for different PG muropeptides, suggesting that PG muropeptides for autoantibody investigation and immune signaling are too complex to be represented by minimal bioactive PG fragments such as MDP, iE-DAP, and N-glycolyl MDP (ng-MDP).