title: Evidence for the modulation of Pseudomonas aeruginosa exotoxin A-induced pore formation by membrane surface charge density.
abstract: 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 micron 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 (Kd = 2-8 microM; 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 pKa 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Ontario, Canada.
authors.forename: D M
authors.initials: DM
authors.lastname: Rasper
authors.forename: A R
authors.initials: AR
authors.lastname: Merrill
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D002463:Cell Membrane Permeability; D018699:Coated Vesicles / Q000737:chemistry; D004926:Escherichia coli / Q000166:cytology; D005098:Exotoxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry*; D016062:Porosity / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D005452:Fluoresceins; D007202:Indicators and Reagents; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; C007740:fluorexon