Sagilkumar, A. C. | bioRxiv Science

bioRxiv2026

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.

microbiology↗

bioRxiv2025

Sorafenib, a clinically approved kinase inhibitor attenuates Streptococcus pneumoniae pathogenesis in vivo by targeting serine/threonine kinase StkP

Streptococcus pneumoniae is a respiratory commensal bacterium responsible for over one million annual fatalities globally, particularly among children under five years of age. The rapid emergence of macrolide-resistant strains led the WHO in 2024 to designate S. pneumoniae as a priority pathogen, underscoring the need for alternative strategies such as anti-virulence therapy. Here, we repurposed the FDA-approved cancer drug, sorafenib identified by in silico screening of compounds targeting the bacterial Serine/Threonine kinase protein StkP, an essential regulator of cell division and peptidoglycan synthesis conserved across many bacterial pathogens. Sorafenib interacts with the StkP-kinase domain and exhibited broad-spectrum activity against diverse pneumococcal serotypes including multi-drug-resistant clinical isolates. Ectopic expression of StkP in both wild-type and isogenic mutant strains conferred partial resistance to sorafenib, confirming on-target activity. Scanning electron microscopy revealed aberrant cell-wall morphology, and differential viability staining demonstrated increased membrane permeability. Consistently, sorafenib-treated bacteria showed significantly higher complement C3 deposition and consequent killing in human serum. In human lung epithelial cells, sorafenib reduced bacterial adherence and invasion without detectable host cytotoxicity. Serial passaging at sub-microbicidal concentrations indicated a low propensity for resistance development in vitro. In vivo, sorafenib administration reduced mortality and lung bacterial load in a mouse model of pneumonia. Taken together, our data identify StkP as target of sorafenib in S. pneumoniae and justify its continued preclinical development for therapeutic intervention.

microbiology↗