Lipid headgroup composition and pH modulate the cholesterol requirement for pneumolysin activity

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins produced by numerous pathogenic bacteria and represent important virulence factors. CDCs share a conserved structural organization and require membrane cholesterol for pore formation. Although the molecular mechanism of pore formation has been extensively characterized, the cholesterol requirement in modulating CDCs activity remains incompletely understood. Using tethered bilayer lipid membranes (tBLMs) and electrochemical impedance spectroscopy, we found that lower cholesterol concentrations were sufficient to support PLY-mediated membrane disruption in membranes containing lipids with less hydrated headgroups. In contrast to the reduced activity of PLY on human erythrocytes at acidic pH, substantial PLY-mediated damage was observed in tBLMs under acidic conditions, accompanied by a reduced cholesterol requirement. Whether this effect results from pH-dependent changes in PLY itself or in the physicochemical properties of membrane lipids remains to be determined. Finally, temperature did not alter the cholesterol threshold for PLY activity but enhanced membrane disruption, likely by reducing membrane viscosity. Although tBLMs do not fully recapitulate the complexity of biological membranes, their well-defined composition and structural properties make them a valuable platform for dissecting protein-membrane interactions that are difficult to resolve in cellular systems. These findings identify membrane composition and environmental conditions as key determinants of the cholesterol requirement for PLY activity.