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 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 demonstrate that both PLY and membrane cholesterol promote vesicle-membrane interactions. Characterization 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, but not the toxoid mutant PLYW433F fuse with human immune cells, delivering toxin and causing membrane damage. Together our results reveal a noncanonical, pore-independent mode of toxin dissemination, in which, vesicle-bound pneumolysin fuses and destabilize target cell membranes, representing a new proposal for EV-mediated toxin activity and a potential target for therapeutic intervention.