Drosocin, pyrrhocoricin, and apidaecin, representing the short (18−20 amino acid residues)
proline-rich antibacterial peptide family, originally isolated from insects, were shown to act on a target
bacterial protein in a stereospecific manner. Native pyrrhocoricin and one of its analogues designed for
this purpose protect mice from bacterial challenge and, therefore, may represent alternatives to existing
antimicrobial drugs. Furthermore, this mode of action can be a basis for the design of a completely novel
set of antibacterial compounds, peptidic or peptidomimetic, if the interacting bacterial biopolymers are
known. Recently, apidaecin was shown to enter Escherichia coli and subsequently kill bacteria through
sequential interactions with diverse target macromolecules. In this paper report, we used biotin- and
fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin analogues to identify biopolymers that bind to
these peptides and are potentially involved in the above-mentioned multistep killing process. Through
use of a biotin-labeled pyrrhocoricin analogue, we isolated two interacting proteins from E. coli. According
to mass spectrometry, Western blot, and fluorescence polarization, the short, proline-rich peptides bound
to DnaK, the 70-kDa bacterial heat shock protein, both in solution and on the solid-phase. GroEL, the
60-kDa chaperonin, also bound in solution. Control experiments with an unrelated labeled peptide showed
that while binding to DnaK was specific for the antibacterial peptides, binding to GroEL was not specific
for these insect sequences. The killing of bacteria and DnaK binding are related events, as an inactive
pyrrhocoricin analogue made of all-d-amino acids failed to bind. The pharmaceutical potential of the
insect antibacterial peptides is underscored by the fact that pyrrhocoricin did not bind to Hsp70, the human
equivalent of DnaK. Competition assay with unlabeled pyrrhocoricin indicated differences in GroEL and
DnaK binding and a probable two-site interaction with DnaK. In addition, all three antibacterial peptides
strongly interacted with two bacterial lipopolysaccharide (LPS) preparations in solution, indicating that
the initial step of the bacterial killing cascade proceeds through LPS-mediated cell entry.