Mutational analysis of the role of charged residues in target-cell binding, potency and specificity of the pediocin-like bacteriocin sakacin P

Abstract

The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P. Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction. All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2-15-fold reduction in potency. The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T). This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance. Somewhat more deleterious was the removal of positive charges at the N-and C-terminal ends (K1T, K43T). Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins. Mutated peptides in which the net positive charge was increased by one were also constructed. Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e. an extra positive charge at the Cterminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e. an extra positive charge at the N-terminus) had reduced potency. Sakacin P contains only one negatively charged residue (Asp17). This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious. Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic. Thus, charged residues in the N-terminal half may -apparently via the initial electrostatic binding of the bacteriocin to target cells -influence the target-cell specificity.Keywords : antimicrobial peptides, mutagenesis, lactic acid bacteria INTRODUCTIONMany Gram-positive bacteria produce antimicrobial peptides, generally termed bacteriocins. These peptides are usually cationic, less than 50 amino acid residues long, contain an amphiphilic or hydrophobic region, and often kill their target cells by permeabilizing the cell membrane (Nes et al., 1996(Nes et al., , 2002. Antimicrobial peptides with these characteristics are also produced by plants and a wide variety of animals, including humans, and are thus widely distributed in nature M. Kazazic, J. Nissen-Meyer and G. Fimland phospholipid-containing membranes and\or acidic cell walls, whereas their amphiphilic\hydrophobic character enables membrane permeabilization. The pediocin-like bacteriocins constitute a dominant and well-studie...

Mutational Analysis of Mesentericin Y105, an Anti- Listeria Bacteriocin, for Determination of Impact on Bactericidal Activity, In Vitro Secondary Structure, and Membrane Interaction

“…Its helix content would be sufficient to account for both its low biological activity and the emission shift in fluorescence. The G13E substitution introduces a negative charge in the positive segment of the N-terminal half of the bacteriocins that is proposed to be involved in the primary binding of the peptide to the membrane (8,34).…”

“…It is generally observed that mutations are deleterious for bactericidal activity, but in some cases, they reinforce the antagonist potency (18,34,41). All these "enhancing" substitutions involve an increase in the net positive charge, H12K, T20K, and 44K in sakacin P (34). Binding of pediocin PA-1, studied by tryptophan fluorescence (7), showed that the K11L-H12L derivative bound weakly to the lipid vesicles.…”

“…In attempts to elucidate the structure-function relationships of class II bacteriocins, many studies analyzed either the bio-logical activity (5,11,18,19,21,34,41,47) or the in vitro binding to phospholipid vesicles (7) of bacteriocin derivatives. It is generally observed that mutations are deleterious for bactericidal activity, but in some cases, they reinforce the antagonist potency (18,34,41).…”

1.6-Å Crystal Structure of EntA-im

“…These two domains are separated by a flexible hinge that enables the two domains to move relative to each other. The N-terminal domain binds to the target cell surface (10,11), whereas the more hydrophobic C-terminal domain enters the hydrophobic part of the cell membrane (3,12,13). Despite their extensive sequence similarity, the pediocin-like bacteriocins differ markedly with respect to the bacteria they kill (6,14).…”

Produção de bacteriocina por Bifidobacterium lactis a partir de soro de leite

“…Much effort has been devoted to the subclass IIA of bacteriocins to determine the structureefunction relationships. Though variants generated in these types of studies are useful from an academic standpoint, none of them display increased activity against several microorganisms (Kazazic, Nissen-Meyer, & Fimland, 2002).…”