Basis for Selectivity of Cationic Antimicrobial Peptides for Bacterial Versus Mammalian Membranes

Abstract

Novel cationic antimicrobial peptides typified by structures such as KKKKKKAAXAAWAAXAA-NH 2 , where X ‫؍‬ Phe/Trp, and several of their analogues display high activity against a variety of bacteria but exhibit no hemolytic activity even at high dose levels in mammalian erythrocytes. To elucidate their mechanism of action and source of selectivity for bacterial membranes, phospholipid mixtures mimicking the compositions of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids ؉ cholesterol) were challenged with the peptides. We found that peptides readily inserted into bacterial lipid mixtures, although no insertion was detected in model "mammalian" membranes. The depth of peptide insertion into model bacterial membranes was estimated by Trp fluorescence quenching using doxyl groups variably positioned along the phospholipid acyl chains. Peptide antimicrobial activity generally increased with increasing depth of peptide insertion. The overall results, in conjunction with molecular modeling, support an initial electrostatic interaction step in which bacterial membranes attract and bind peptide dimers onto the bacterial surface, followed by the "sinking" of the hydrophobic core segment to a peptide sequence-dependent depth of ϳ2.5-8 Å into the membrane, largely parallel to the membrane surface. Antimicrobial activity was likely enhanced by the fact that the peptide sequences contain AXXXA sequence motifs, which promote their dimerization, and possibly higher oligomerization, as assessed by SDS-polyacrylamide gel analysis and fluorescence resonance energy transfer experiments. The high selectivity of these peptides for nonmammalian membranes, combined with their activity toward a wide spectrum of Gram-negative and Gram-positive bacteria and yeast, while retaining water solubility, represent significant advantages of this class of peptides.

Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

“…20 In the present work, all 2-, 3-, and Leu isomeric peptides migrate on SDS-PAGE as dimers. Such dimerization must relate to the presence of highly potent helix-helix interaction motifs in their sequence.…”

“…A similar pattern was noted earlier for Lys-tagged 25-residue peptides with 19-residue core hydrophobic segments. 17 However, when this paradigm was applied to zwitterionic mammalian-like membranes, we found for similar peptides containing highly charged Lys tags attached to a core hydrophobic sequence, 20 that peptides ultimately cannot leave the bulk water for attachment/insertion into erythrocyte-like bilayers until their CSH begins to approach sufficiently high levels, as in the case where the sequence contains at least two Ala-toLeu substitutions (see Fig. 2).…”

“…19 In addition, while the majority of peptides from the ''shorter'' series showed absence of toxicity to mammalian cells, substitution of four core Ala residues to more hydrophobic residues caused appearance of significant hemolytic activity in erythrocytes, suggesting that hydrophobic character became elevated to a level where partial insertion into zwitterionic bilayers that mimic the outer leaflet of mammalian plasma membrane composition becomes possible. 20 Peptide 6K-F17 (KKKKKKAAFAAWAAFAA-NH 2 ) from the ''shorter'' series with six Lys residues at the N-terminus showed the best characteristics among the family-the highest antimicrobial activity along with negligible hemolytic activity. 16,19 Since these totally synthetic nonamphipathic core peptides were found to be active in the low-medium micromolar range, and show little target or L-vs. D-residue specificity 16 -similarly to the majority of gene-encoded CAPs-their microbial and mammalian cell membrane disruption can likely be ascribed, at least in large measure, to one of the characterized nonstereospecific mechanisms described above.…”

Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions

“…In designing this peptide, Ala was chosen as the background residue due to its midrange hydrophobicity and frequent occurrence in membrane domains. As well, the AxxxA ("small-xxxsmall") sequence motif promotes peptide dimerization in membranes (9,10), which has been suggested to enhance antimicrobial activity (11). The Trp residue in the hydrophobic core serves as the fluorescent probe, whereas the positively charged Lys residues were included at the terminus to maintain cationic properties and enhance peptide solubility.…”

Biomimetic Model Membrane Systems Serve as Increasingly Valuable in Vitro Tools

“…The negatively charged bacterial lipid PG behaved in a similar manner to the PS, emphasizing the importance of an electrostatic interaction with the positively charged AMP (Matsuzaki et al, 1989;Lohner and Prenner, 1999). A more accurate representation of the E. coli membrane was provided by using a headgroup ratio of 75:20:5 POPE/DPPG/CL in SUVs and LUVs (Glukhov et al, 2005) to study the differential targeting of novel cationic AMPs to bacterial versus mammalian membranes. They found that the initial attraction between the positive AMP and negatively charged bacterial membranes was an important component of bacterial/mammalian selectivity.…”