EVIDENCE FOR THE MODULATION OF PSEUDOMONAS-AERUGINOSA EXOTOXIN A-INDUCED PORE FORMATION BY MEMBRANE-SURFACE CHARGE-DENSITY

The lipid requirement for the binding of wild-type Pseudomonas aeruginosa exotoxin A (ETA) to model endosomal membrane vesicles was evaluated using a fluorescence quenching technique. The binding of toxin to monodisperse model membrane vesicles (0.1 mu m diameter) composed of various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) prepared by an extrusion method [Hope, M. J., et al. (1986) Chem. Phys. Lipids 40 89-107] was pH-dependent, with maximal binding observed at pH 4.0. Analysis of the binding curves indicated that the interaction of ETA with the membrane bilayer is dominated by a set of high-affinity binding sites (K-d 2-8 mu M; 60:40 (mol:mol) POPC/POPS large unilamellar vesicles (LUV)). The binding of toxin to membrane vesicles was highly pH-dependent, but was ionic strength-independent. Toxin-induced pore formation in the lipid bilayer, as measured by the release of the fluorescent dye, calcein, from LUV was pH-dependent, with optimal dye release occurring at pH 4.0. The rate of dye release from membrane vesicles decreased rapidly with increasing pH and approached zero at pH 6.0 and higher. The pK(a) for this process ranged over 4.3-4.5. Calcein release from LUV was also sensitive to changes in the ionic strength of the assay buffer, with maximal release occurring at 50 mM NaCl. Higher ionic strength medium resulted in a dramatic reduction in the rate of dye release from vesicles, indicating that the toxin-induced pore is modulated by ionic interactions. Further evidence for the role of electrostatic interactions between toxin and the membrane was provided by the effect of POPS on the kinetic properties of the pore. Maximal dye release (pH 4.0) was observed for vesicles composed of 60 mol % POPS. At higher vesicle POPS content (i.e., 100 mol %), the rate of dye release was reduced by 6-fold compared with 60 mol % POPS and 2-fold compared with 20 mol % POPS. The toxin-induced membrane permeabilization was temperature-dependent, with an activation energy (E(a)) near 14 kcal/mol (59 kJ/mol). A break point in the Arrhenius plot for toxin-induced pore activity indicated that the permeabilization process was sensitive to the physical state of the membrane bilayer.