J. Biol. Chem., Vol. 261, Issue 17, 7871-7878, Jun, 1986
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
W Boisvert, KS Cheung, SA Lerner and M Johnston
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.
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