Membrane interactions of designed cationic antimicrobial peptides: The two thresholds

Abstract

ABSTRACT:Novel cationic antimicrobial peptides (CAPs) designed in our lab-typified by sequences such as KKKKKKAAX-AAXAAXAA-NH 2 , where X 5 Phe/Trp-display high antibacterial activity but exhibit little or no hemolytic activity towards human red blood cells even at high doses.To clarify the mechanism of their selectivity for bacterial versus mammalian membranes and to increase our understanding of the relationships between primary sequence and bioactivity, a library of derivatives was prepared by increasing segmental hydrophobicity, in which systematic substitutions of Ala for two, three, or four Leu residues were made. Conformationally constrained dimeric and cyclic derivatives were also synthesized. The peptides were examined for activity against pathogenic bacteria (Pseudomonas aeruginosa),

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

“…The F17 sequence contains three AxxxA motifs (two AAFAA sequences and a central AAWAA sequence); the F17-4L sequence retains the central motif (ALWLA). Consistent with our previous findings (11,14), the MW exp /MW theor values for both 6K-F17 and 6K-F17-4L are 2.4 ( Fig. 2), indicating that these two CAPs form SDS-resistant dimers on SDS-polyacrylamide gels, even at the low loading levels used in silver stain analysis.…”

“…In striking contrast, peptides with high hydrophobicity (3K-F17-4L-3K and 6K-F17-4L) showed hemolytic activities at every concentration tested. In parallel to the mammalian membrane-disruptive effects reported above, these results support the notion that CAPs with greater hydrophobicity are prone to inducing hemolysis against human erythrocytes (14). The 6K-F17 peptide did display a detectable level of hemolysis at the highest concentration tested (2.7 Ϯ 1.2% at 320 M); the all-D chiral version of this peptide displayed no hemolysis at 325 M (14).…”

“…CAP Interactions with Model Bacterial Membranes-Because the core segment hydrophobicity of CAPs is a key factor that affects bacterial membrane insertion directly and antimicrobial activity indirectly (11,14), it becomes crucial to understand how peptide hydrophobicity influences their membranedisruptive ability, particularly where host membranes are at stake. To address this, we compared the mechanistic details of the interactions of two CAPs with contrasting core hydrophobicities (6K-F17 with a "low" core segment hydrophobicity (1.48) and 6K-F17-4L with a "high" core segment hydrophobicity (3.14)) (Table 1) in a bacterial membrane lipid bilayer model using a combination of "simultaneous" AFM and ATR-FTIR techniques.…”

“…On the basis of the highly active CAP 6K-F17, we went on to design a series of CAPs with a range of hydrophobicity values and observed a "threshold hydrophobicity" for selective bacterial membrane insertion (11)(12)(13). However, once the core segment hydrophobicity of the CAPs is beyond an upper threshold, as in the case in which the sequence contains two or more Ala-to-Leu substitutions, the CAPs generally have reduced antimicrobial activity and display increased toxicity to mammalian membranes (14). In agreement with these findings, other workers confirmed the importance of peptide hydrophobicity in membrane selectivity and insertion and for antimicrobial activity (15,16).…”

“…Hemolytic Activity-The hemolytic toxicity levels of the designed CAPs were measured in human RBCs as described previously (14). Freshly collected venous human blood with heparin was centrifuged at 1000 ϫ g for 5 min at 11°C to remove the buffy coat, and the RBCs obtained were washed three times with PBS.…”