Mechanisms of action of chloroalanyl antibacterial peptides. Identification of the intracellular enzymes inactivated on treatment of Escherichia coli JSR-O with the dipeptide beta Cl-LAla-beta Cl-LAla.
The dipeptide beta Cl-LAla-beta Cl-LAla is an antibacterial agent designed to utilize bacterial peptide transport for intracellular delivery of the alanine racemase inactivator beta Cl-LAla. The minimum inhibitory concentrations (MICs) for the peptide against Gram-negative species grown on enriched agar medium range from 1.56 to 12.5 micrograms/ml; MICs are increased to greater than 100 micrograms/ml when D-alanine is included in the medium, indicating that alanine racemase is, in fact, inhibited in sensitive species. When susceptible Gram-negative cells are grown on a minimal medium, D-alanine supplementation alone does not increase the MICs for beta Cl-LAla-beta Cl-LAla, but complete protection is afforded by supplementation with D-alanine, L-valine, L-leucine, and L-isoleucine. In liquid culture, the peptide is: bactericidal and lytic against Escherichia coli JSR-O growing in enriched medium or in minimal medium supplemented with the branched-chain amino acids; only inhibitory against these cells growing in minimal medium supplemented with D-alanine; and ineffective against these cells in minimal medium containing the branched-chain amino acids plus D-alanine. Cells exposed to beta Cl-LAla-beta Cl-LAla (with the protection of the four amino acids) have specific activities of both alanine racemase and transaminase B that are lower than those of cultures not treated with the peptide. Finally, E. coli JSR-O alanine racemase experiences time-dependent loss of activity when exposed to the dipeptide in the presence of aminopeptidases; the dipeptide alone is not an inactivator of the racemase in vitro. These results suggest the following mechanism of action for beta Cl-LAla-beta Cl-LAla: transport of the dipeptide into the cell; intracellular hydrolysis to give accumulation of beta Cl-LAla; and subsequent inactivation of targeted enzymes. Whether inactivation of the racemase or of the transaminase determines the pathophysiologic effects of the peptide depends on the composition of the growth medium.
D000411:Alanine Racemase / Q000302:isolation & purification / Q000378:metabolism*; D000591:Amino Acid Isomerases / Q000378:metabolism*; D000596:Amino Acids / Q000494:pharmacology; D002701:Chloramphenicol / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D007202:Indicators and Reagents; D008826:Microbial Sensitivity Tests; D009842:Oligopeptides / Q000494:pharmacology
D000596:Amino Acids; D004151:Dipeptides; D007202:Indicators and Reagents; D009842:Oligopeptides; C049303:beta-chloroalanyl-beta-chloroalanyl-beta-chloroalanine; D002701:Chloramphenicol; C049302:beta-chloroalanyl-beta-chloroalanine; D000591:Amino Acid Isomerases; D000411:Alanine Racemase