{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T10:45:50Z","timestamp":1791456350540,"version":"4.3.4"},"reference-count":38,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J of Extracellular Vesicle"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Pneumolysin (PLY) is a cholesterol\u2010dependent pore\u2010forming toxin and a key virulence factor of\n                    <jats:italic>Streptococcus pneumoniae<\/jats:italic>\n                    , the leading cause of pneumonia worldwide in children aged below 5 years. At sublytic toxin doses, host cells shed PLY\u2010laden extracellular vesicles (EVs) during the membrane repair response. However, it remains unclear how these toxin\u2010bearing EVs engage and damage target cell membranes. Here, we combine molecular dynamics simulations, liposome fusion assays and cell\u2010based experiments to elucidate the membrane interaction potential of vesicle\u2010bound PLY. Simulations indicate that EV\u2010embedded 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\u2010EM analysis demonstrate that both PLY and membrane cholesterol promote vesicle\u2010membrane interactions. Characterisation of vesicle subpopulations released from PLY\u2010challenged monocytes by western blotting and immunogold electron microscopy demonstrated that plasma membrane\u2010derived microvesicles are preferentially enriched in membrane\u2010bound PLY compared to small extracellular vesicles. Consistent with these findings, MVs purified from wild\u2010type PLY\u2010challenged 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\n                    <jats:sup>+<\/jats:sup>\n                    T cells showed higher PLY positivity upon MV co\u2010incubation and higher cell death compared to monocytes at same dose. Together, our results reveal a noncanonical mode of toxin dissemination, in which vesicle\u2010bound pneumolysin fuses and destabilises target cell membranes, representing an alternative pathway for EV\u2010mediated toxin activity and a potential target for therapeutic intervention.\n                  <\/jats:p>","DOI":"10.1002\/jev2.70387","type":"journal-article","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T17:46:29Z","timestamp":1790790389000},"source":"Crossref","is-referenced-by-count":1,"title":["Extracellular Vesicle\u2010Bound Bacterial Toxin Pneumolysin Triggers Membrane Engagement and Damage Beyond Canonical Pore Formation"],"prefix":"10.1002","volume":"15","author":[{"given":"Aswathy C.","family":"Sagilkumar","sequence":"first","affiliation":[{"name":"Host\u2010Pathogen Laboratory Pathogen Biology Division BRIC\u2010Rajiv Gandhi Centre for Biotechnology  Thiruvananthapuram India"},{"name":"Regional Centre for Biotechnology  Faridabad India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-8049-9580","authenticated-orcid":false,"given":"Avinashi Lal","family":"Kushwaha","sequence":"additional","affiliation":[{"name":"Host\u2010Pathogen Laboratory Pathogen Biology Division BRIC\u2010Rajiv Gandhi Centre for Biotechnology  Thiruvananthapuram India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anushka","family":"Shitut","sequence":"additional","affiliation":[{"name":"Tata Institute of Fundamental Research Hyderabad  Telangana India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dheeraj Kumar","family":"Sarkar","sequence":"additional","affiliation":[{"name":"Tata Institute of Fundamental Research Hyderabad  Telangana India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sruthika","family":"Sukumar","sequence":"additional","affiliation":[{"name":"Host\u2010Pathogen Laboratory Pathogen Biology Division BRIC\u2010Rajiv Gandhi Centre for Biotechnology  Thiruvananthapuram India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Subhajit","family":"Mahanty","sequence":"additional","affiliation":[{"name":"Molecular Biophysics Unit Indian Institute of Science  Bengaluru India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3181-0108","authenticated-orcid":false,"given":"Somnath","family":"Dutta","sequence":"additional","affiliation":[{"name":"Molecular Biophysics Unit Indian Institute of Science  Bengaluru India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1090-5199","authenticated-orcid":false,"given":"Jagannath","family":"Mondal","sequence":"additional","affiliation":[{"name":"Tata Institute of Fundamental Research Hyderabad  Telangana India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4381-5037","authenticated-orcid":false,"given":"Karthik","family":"Subramanian","sequence":"additional","affiliation":[{"name":"Host\u2010Pathogen Laboratory Pathogen Biology Division BRIC\u2010Rajiv Gandhi Centre for Biotechnology  Thiruvananthapuram India"},{"name":"Regional Centre for Biotechnology  Faridabad India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,30]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High Performance Molecular Simulations Through Multi\u2010Level Parallelism From Laptops to Supercomputers","volume":"1","author":"Abraham M. 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