Physicochemical characterization and structure function relationship for the interaction of pediocin PA-1 with Listeria monocytogenes cells and lipid vesicles

The application of the bacteriocin pediocin PA-1 as a preservative is covered by several U.S. and European patents. This dissertation investigated pediocin PA-1 binding and pore formation in target membranes, and examined the structure function relations for pediocin PA-1 interactions with model membranes.; Pediocin PA-1 (80 AU/ml) reduced L. monocytogenes Scott A viability by 4.6-log cycles and caused concomitant efflux of intracellular K{dollar}sp{lcub}+{rcub}{dollar} and Pi, and depletion of cytoplasmic ATP in a concentration- and time-dependent fashion. These results confirmed that the target for pediocin PA-1 action is the cytoplasmic membrane. Pediocin PA-1 caused the time- and concentration-dependent release of entrapped carboxyfluorescein (CF) from Listeria lipid vesicles with an apparent binding constant of {dollar}1.4times 10sp7{dollar} M{dollar}sp{lcub}-1{rcub}{dollar} at pH 6.0. CF efflux rates increased with more acidic pH, by the imposition of a {dollar}Deltasb{lcub}Psi{rcub},{dollar} and in the absence of salt. Pediocin PA-1 also permeablized pure phospholipid vesicles. This demonstrates that, pediocin PA-1 functioned in the absence of a protein receptor and the formation of pore complexes was a {dollar}Deltasb{lcub}Psi{rcub}{dollar}-enhanced process.; Binding of pediocin PA-1 and four synthetic pediocin fragments was examined using tryptophan fluorescence. Binding of the whole peptide and the 15-amino-acid N-terminal fragment to phosphatidylglycerol vesicles caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constant for acrylamide quenching of pediocin PA-1 fluorescence in buffer was more than twice the value in lipid vesicles, suggesting insertion of tryptophan residues into the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively-charged amino acid residues was present, but bound weakly when this patch was mutated out. Quantitative comparison of changes in fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the anionic lipid vesicles paralleled the peptide's net positive charge. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Increasing the anionic phospholipid content of vesicles resulted in stronger pediocin PA-1 binding. These results demonstrate that electrostatic interactions govern pediocin PA-1 binding. Various factors influence pediocin PA-1 activity through their effects on electrostatic interactions. Results from this present study will facilitate rational and expanded applications of pediocin PA-2 to ensure food safety which depend on multiple-hurdle preservation systems including bacteriocins as a vital part.