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 aerugin osa exotoxin A (ETA) to model endosomal membrane vesicles was evaluate d using a fluorescence quenching technique. The binding of toxin to mo nodisperse model membrane vesicles (0.1 mu m diameter) composed of var ious molar ratios of almitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholin e (POPC) and palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) prepared by an extrusion method [Hope, M. J., et al. (1986) Chem. Phy s. Lipids 40 89-107] was pH-dependent, with maximal binding observed a t pH 4.0. Analysis of the binding curves indicated that the interactio n of ETA with the membrane bilayer is dominated by a set of high-affin ity binding sites (K-d 2-8 mu M; 60:40 (mol:mol) POPC/POPS large unila mellar 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 th e fluorescent dye, calcein, from LUV was pH-dependent, with optimal dy e release occurring at pH 4.0. The rate of dye release from membrane v esicles 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. Calce in release from LUV was also sensitive to changes in the ionic strengt h of the assay buffer, with maximal release occurring at 50 mM NaCl. H igher ionic strength medium resulted in a dramatic reduction in the ra te of dye release from vesicles, indicating that the toxin-induced por e is modulated by ionic interactions. Further evidence for the role of electrostatic interactions between toxin and the membrane was provide d 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 % P OPS. 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 permeabilizati on 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-i nduced pore activity indicated that the permeabilization process was s ensitive to the physical state of the membrane bilayer.