PG-BIR · Elucidating the Role of Peptidoglycan Fragments in β-Lactam-Induced Host Immune Responses

β-Lactam antibiotics constitute roughly half of clinical use and act by inhibiting peptidoglycan (PG) biosynthesis, causing lysis. The resulting PG fragments can traverse the small-intestinal epithelium and activate NOD1/NOD2–NF-κB signalling, yet the scale and consequences of β-lactam–induced PG release—particularly in multidrug-resistant (MDR) versus susceptible bacteria—remain poorly defined. PG-βIR will delineate how clinically used β-lactams modulate PG-fragment outputs and host responses. I will deploy small-intestinal apical-out organoids and a small-intestine organ-on-chip to quantify transepithelial PG flux by LC–MS/MS muropeptidomics, epithelial signalling (NF-κB readouts, cytokines) and barrier function, and we will test mechanistic requirements using organoids from knockout lines in epithelial transport and innate recognition (SLC15A1, SLC46A2, RNF43, NOD1, NOD2, IL-1Ra). Comparative panels will include resistant and susceptible Escherichia coli and Enterococcus faecalis; the most pro-inflammatory resistant E. coli identified ex vivo will progress to in-vivo validation. In-vivo confirmation will use gnotobiotic mice under β-lactam exposure, with compartment-resolved PG profiles (lumen, tissue, serum), NF-κB activation and cytokines analysed under a pre-specified plan. The programme is organoid-first to minimise animal use; in-vivo work confirms key mechanisms. Expected outputs are a comparative atlas of β-lactam-induced PG outputs in MDR versus susceptible backgrounds, a mechanistic map of epithelial transport/sensing for PG translocation, and an inflammatory signature linking PG species to NOD1/NOD2 responses. These decision-grade results will inform antimicrobial stewardship (integrating inflammatory risk) and guide safer β-lactam regimens and next-generation dual-action prodrugs; data and protocols will follow FAIR, with open LC–MS/MS datasets and organoid/organ-on-chip methods.