cecropin-p1-li and Hemolysis

Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH(2) terminus of the peptide and the fragment arasin 1(1-23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1-23) were shown to be non-toxic to human red blood cells and arasin 1(1-23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1-23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1-23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1-23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1-23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Anti-Infective Agents; Antifungal Agents; Antimicrobial Cationic Peptides; Cell Membrane Permeability; Chitin; Circular Dichroism; Escherichia coli; Hemolysis; Humans; Kinetics; Microbial Sensitivity Tests; Microbial Viability; Molecular Sequence Data; Peptide Fragments; Peptides; Proline-Rich Protein Domains; Structure-Activity Relationship

Two antimicrobial peptides, cecropin P1 (CP1), with a C-terminal carboxyl group, and PR-39, with an amidated, C-terminus, are found in the small intestine of the pig. Each is active against both Gram-positive and Gram-negative bacteria. We have synthesized these peptides and several analogs, including the D-enantiomers and the retro sequences, each with a free or acetylated amino terminus. The CP1 amide was also prepared. The retro CP1 peptides were much less active than the parent CP1 peptide, confirming the importance of sequence or the amide bond and helix dipole direction, and the N alpha-acetyl peptides were also less active, indicating that a free amino terminus is essential for high activity. The ratio of the lethal concentration of L/D isomers of CP1 is less than 1 for Gram-negative, but greater than 1 for Gram-positive bacteria. PR-39 showed no significant chiral selectivity toward Escherichia coli, Bacillus subtilis and Streptococcus pyogenes, but the L/D ratio was high for Pseudomonas aeruginosa (66), and very high for Staphylococcus aureus (> 1000). In the latter case the lethal concentration for the D-isomer was 0.57 microM, whereas this organism was quite resistant to the L-isomer (> 600 microM). Thus the enantiomers of CP1 and PR-39 are not equally active for all species. In a plate assay with a very small log-phase inoculum of Staph aureus, D-PR-39 produced a clear zone of killing surrounded by a zone of stimulated growth. After prolonged incubation the two zones became one clear zone. Addition of D-PR-39 to the wells of a dense turbid plate of growing cells showed a cleared zone for each of the test organisms, indicating that PR-39 lyses the bacteria rather than simply inhibiting their multiplication.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Arginine; Circular Dichroism; Hemolysis; Intestines; Molecular Sequence Data; Peptides; Proline; Sheep; Structure-Activity Relationship; Swine