Bacterial Membrane Vesicles Derived from Probiotic B. Subtilis Promote Wound Re-Epithelialization through Engineered Delivery of NOD2 Ligands

Bacterial membrane vesicles (MVs) are bilipid nanoparticles secreted as a conserved method of intercellular communication. MVs from Gram-positive bacteria carry diverse bacterial products and represent a unique opportunity in the growing field of postbiotics. This native machinery for mediating microbe-host interactions makes MVs a promising tool for intestinal drug delivery in the context of Crohn’s disease (CD). CD is a chronic inflammatory bowel disease characterized by relapsing inflammation of the digestive tract. CD is associated with (1) loss of function mutations in the host gene encoding the nuclear-binding oligomerization domain 2 (NOD2) pattern recognition receptor and (2) a gut microbiome with reduced capabilities to generate NOD2 stimulating muropeptides from peptidoglycan. However, there are currently no therapeutic strategies targeting NOD2 for CD management. Here, we demonstrate for the first time that MVs from B. subtilis can deliver NOD2 ligands in vitro. Using HCT116 cells, a human colorectal carcinoma cell line, we demonstrate that treatment with isolated MVs induces IL8 secretion in a NOD2-dependent manner. Furthermore, MVs from B. subtilis promote re-epithelialization after in vitro scratch wounding. This effect was completely blunted by the addition of an inhibitor for RIPK2, a downstream transducer required for NOD2 signaling. Currently, we are applying metabolic engineering of peptidoglycan degradation and recycling pathways to optimize muropeptide packaging in B. subtilis MVs. This work suggests that facilitated delivery of muropeptides by probiotic-derived MVs is a promising strategy to enhance NOD2 signalling activity and promote reconstitution of the epithelial barrier during inflammation-induced injury.