Pores formed by cholesterol-dependent cytolysins (CDCs), such as Streptolysin O (SLO) and Perfringolysin O (PFO), are critical for bacterial pathogenesis. However, studies to date have primarily relied on synthetic lipids with artificially high cholesterol content, limiting insights into pore formation on physiological membranes. Additionally, bulk ensemble techniques used in these studies preclude single-molecule resolution of kinetic intermediates. While recent atomic force microscopy (AFM) and single-molecule experiments revealed pore assembly pathways on synthetic systems, a key question remains: How does pore dynamics differ between artificial and natural mammalian plasma membranes?
To address this, we engineered HIV virus-like particles (VLPs) encapsulating GFP, as a proxy for liposomes made directly from the plasma membrane of mammalian cells. Using single-molecule fluorescence microscopy, we compared pre-pore assembly and pore insertion dynamics between these VLPs and synthetic liposomes. Nucleation and polymerization of SLO and PFO are significantly accelerated on mammalian membranes compared to synthetic counterparts. Despite this, pore insertion rates remain identical between systems, indicating membrane composition selectively impacts pre-pore assembly but not final insertion steps.
Mechanistically, we identified a critical long-lived single-molecule binding state which is enriched on mammalian membranes. These rare, stable interactions fuel the nucleation and polymerization of functional oligomers, enabling faster pre-pore formation without altering insertion kinetics. This work highlights the complexity of CDC interactions with membranes, providing a framework for exploring how different lipid compositions impact pore-formation kinetics.