Protease-induced activation of colonic afferent neurons by a gut bacterium associated with inflammatory bowel disease

Background: Inflammatory bowel disease (IBD) involves relapsing inflammation of the gastrointestinal tract and is frequently associated with abdominal pain, yet effective treatments remain limited. Proteolytic activity is elevated in IBD patients, and proteases can activate visceral pain pathways via protease-activated receptors (PARs). Notably, protease activity from the gut bacterium, Bacteroides vulgatus, has recently been correlated with IBD severity, suggesting a potential microbial contribution to IBD-related pain. We hypothesize that proteases produced by B. vulgatus activate colonic afferent nerves involved in nociception. Methods: B. vulgatus cell-free supernatant (ATCC 8482 strain) or brain heart infusion (BHI) media on nociceptive neuronal activity were examined using ex vivo single-unit colonic afferent recordings from C57Bl/6 mice. Cellular mechanisms were probed by Ca 2 + imaging of dissociated DRG neurons exposed to supernatant superfusion. To specifically assess sensitization, we examined whether exposure of DRG neurons to B. vulgatus supernatant enhanced TRPV1-mediated Ca 2 + influx to capsaicin. A protease inhibitor cocktail (PIC) evaluated protease involvement, and Nav1.8-PAR2 knockout mice determined PAR2 dependence. In vivo visceromotor responses (VMR) to colorectal distension (CRD) were assessed after intracolonic administration of supernatant. Colonic permeability was measured ex vivo via FITC-dextran flux in Ussing chambers. PAR2 N-terminal cleavage by B. vulgatus supernatant was assessed using a fluorescent enzymatic assay. Results: Superfusion of DRG neurons with B. vulgatus supernatant, but not BHI, significantly increased Ca 2+ influx in response to the pro-nociceptive mediator, capsaicin (100 nM; p< 0.0001) by 49%. Ex vivo luminal perfusion of B. vulgatus supernatant, but not BHI, significantly increased spontaneous activity by 50% (p< 0.001) and mechanosensitivity of colonic afferent axons by 48% (p< 0.05). Pre-incubation of B. vulgatus supernatant with a PIC blocked the excitatory effect observed in both bioassays. Additionally, this excitation was suppressed using DRG neurons and colons from Nav1.8 PAR2 knockout mice, whereas it was maintained in the Cre controls. In vivo enema administration of B. vulgatus supernatant significantly increased VMR to CRD by 183% (p< 0.05), whereas consecutive BHI enemas had no effect. Exposure to B. vulgatus supernatant significantly increased colonic mucosal permeability, as indicated by elevated FITC-dextran flux across the colonic epithelium compared with BHI application, an effect blocked using the PIC (p = 0.05). Lastly, PAR2 cleavage was detected in the B. vulgatus supernatant, which was blocked using serine and metalloprotease inhibitors. Conclusion: Proteases secreted by B. vulgatus disrupt permeability and sensitize nociceptive visceral afferent nerves and may be a novel target for pain management in IBD. This work highlights the potential for microbiome-derived proteases to contribute directly to abdominal pain and mucosal injury in IBD patients. Funded by CIHR. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.