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

ABSTRACT 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.