Extracellular Vesicle-Bound Bacterial Toxin Pneumolysin Triggers Membrane Engagement and Damage Beyond Canonical Pore Formation.

Pneumolysin (PLY) is a cholesterol-dependent pore-forming toxin and a key virulence factor of Streptococcus pneumoniae, the leading cause of pneumonia worldwide in children aged below 5 years. At sublytic toxin doses, host cells shed PLY-laden extracellular vesicles (EVs) during the membrane repair response. However, it remains unclear how these toxin-bearing EVs engage and damage target cell membranes. Here, we combine molecular dynamics simulations, liposome fusion assays and cell-based experiments to elucidate the membrane interaction potential of vesicle-bound PLY. Simulations indicate that EV-embedded PLY uses an exposed helix to bind target cell membranes, inducing pronounced curvature, bilayer thinning and water influx. Liposome fusion assays by 3D confocal imaging and Cryo-EM analysis demonstrate that both PLY and membrane cholesterol promote vesicle-membrane interactions. Characterisation of vesicle subpopulations released from PLY-challenged monocytes by western blotting and immunogold electron microscopy demonstrated that plasma membrane-derived microvesicles are preferentially enriched in membrane-bound PLY compared to small extracellular vesicles. Consistent with these findings, MVs purified from wild-type PLY-challenged monocytes, fuse with human peripheral blood mononuclear cells, delivering toxin and causing membrane damage, which was significantly lower with the toxoid mutant, PLYW433F. Among PBMCs, CD4+ T cells showed higher PLY positivity upon MV co-incubation and higher cell death compared to monocytes at same dose. Together, our results reveal a noncanonical mode of toxin dissemination, in which vesicle-bound pneumolysin fuses and destabilises target cell membranes, representing an alternative pathway for EV-mediated toxin activity and a potential target for therapeutic intervention.