Enterococcus-derived cytolysin exacerbates ischemic stroke by disrupting the blood-brain barrier via NLRP3 activation.

BACKGROUND: Ischemic stroke remains a major global health burden. Enterococcus, which is enriched in the gut of stroke patients, produces cytolysin-a pore-forming toxin that disrupts cell membranes. Gut barrier damage may allow such toxins to enter circulation and impair the blood-brain barrier (BBB). In this study, cytolysin-induced BBB disruption via NLRP3 inflammasome activation was investigated with the aim of identifying new therapeutic targets for stroke. METHODS: Ischemic stroke patients were enrolled within 48 h of onset at the First Affiliated Hospital of Wenzhou Medical University. Rectal swabs were collected for 16 S rRNA sequencing and analyzed via QIIME. To investigate the effects of cytolysin-positive Enterococcus on BBB integrity and NLRP3 activation, a photothrombotic stroke model in C57BL/6J mice and in vitro studies with bEnd.3 cells were performed. Cerebral blood flow, BBB permeability, and intestinal barrier function were assessed using established physiological and molecular assays. NLRP3 pathway activation was evaluated by RNA sequencing, qPCR, Western blotting, and ELISA, and its functional role was further confirmed by administration of the selective NLRP3 inhibitor MCC950. Statistical significance was defined as p < 0.05. RESULTS: In a cohort of 100 stroke patients, rectal swabs were collected for 16 S rRNA sequencing and qPCR analysis, and the results identified elevated Enterococcus and cylA gene levels as potential prognostic biomarkers for 360-day mortality. In a photothrombotic stroke mouse model, cytolysin-positive Enterococcus exacerbated stroke severity by increasing BBB permeability. Mechanistically, cytolysin activated nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis, further compromising BBB integrity. Additionally, cytolysin disrupted the intestinal barrier, increased permeability, and may facilitate its entry into the circulation, potentially contributing to BBB damage. Notably, NLRP3 inhibition with MCC950 mitigated neurological deficits and restored BBB integrity. CONCLUSION: Cytolysin aggravates ischemic stroke by compromising the BBB via NLRP3 activation and disrupting the intestinal barrier. Targeting cytolysin and NLRP3 holds promise as a therapeutic strategy for neuroprotection during ischemic stroke.