Selective Lysis of Bacteria but Not Mammalian Cells by Diastereomers of Melittin:  Structure−Function Study

Abstract

Studies on lipid-peptide interactions of cytolytic polypeptides tend to emphasize the importance of the amphipathic alpha-helical structure for their cytolytic activity. In this study, diasetereomers of the bee venom melittin (26 a.a.), a non-cell-selective cytolysin, were synthesized and investigated for their structure and cytolytic activity toward bacteria and mammalian cells. Similarly to the findings with the diastereomers of the less cytolytic peptide pardaxin (33 a.a.) (Shai & Oren. 1996), the melittin diastereomer, lest their alpha-helical structure, which abrogated their hemolytic activity toward human erythrocytes. However, they retained their antibacterial activity and completely lysed both Gram-positive and Gram-negative bacteria, as revealed by transmission electron microscopy. To understand the molecular mechanism underlying this selectivity, binding experiments utilizing the intrinsic tryptophan of melittin, tryptophan quenching experiments using brominated phospholipids, and membrane destabilization studies were done. The data revealed that the melittin diastereomers bound to and destabilized only negatively-charged phospholipid vesicles, in contrast to native melittin, which binds strongly to both negatively-charged and zwitterionic phospholipids. However, the partition coefficient, the depth of penetration into the membrane, and the membrane-permeating activity of the diastereomers with negatively-charged phospholipids were similar to those obtained with melittin. The results obtained do not support the formation of transmembrane pores as the mode of action of the diastereomers, but rather suggest that these peptides bind to the surface of the bacterial membrane, cover it in a "carpet-like" manner, and dissolve it like a detergent. The results presented here together with those obtained with the cytolytic peptide pardaxin suggest that the combination of hydrophobicity and net positive charge may be sufficient in the design of potent diastereomers of antibacterial polypeptides for the treatment of infectious diseases.

Length Effects in Antimicrobial Peptides of the (RW)nSeries

“…The hemolytic activity of model peptides was assessed on fresh sheep erythrocytes (Fitzgerald Inc., Concord, MA). Peptide concentrations yielding 50% hemolysis were used as the hemolytic dose (HD 50 ), determined from dose-response curves (32). The red blood cell suspension was incubated in PBS buffer (pH 7.2) with various volumes of peptide stocks at 37°C for 30 min and then spun down at 3,000 rpm for 10 min.…”

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

“…For example, substitution of four D-amino acids into the melittin B sequences for their L-residues dramatically reduced α-helical structure in 40% TFE, from 73% α-helical content for melittin to 15% for the above D-analog (Oren and Shai, 1997). The advantage of D-amino acid substitutions is D-analog shows negligible cytotoxic effects on mammalian cells and retained the antibacterial activity, while the native melittin B is extremely hemolytic (100% hemolysis at 1 μg/mL).…”

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

“…For most metrics, there was a reduction in cytotoxicity in the order of melittin>PA-1>PA-2>PA-3>PA-4, correlating with reduction in hydrophobicity index (see Table I and reference [42]) but different to the order that antimicrobial activity changes suggesting different modes of membrane perturbation for bacterial and mammalian cells. Altering hydrophobicity, amphipathicity and/or helicity of melittin reduces the cytolytic action of melittin on mammalian membranes [11,18,43,44]. PA-1 and PA-2 are deletion analogues that had 8-fold and 124-fold less hemolytic activity than native melittin [11], are less hydrophobic and demonstrate lower cytotoxicity by MTT assay [42].…”