Synthesis and antibacterial action of cecropin and proline-arginine-rich peptides from pig intestine.

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.