Interaction between Heat Shock Proteins and Antimicrobial Peptides

Abstract

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.

Primary Structure and in Vitro Antibacterial Properties of the Drosophila melanogaster Attacin C Pro-domain

“…Among all the other bacteria (Gram-positive, Gram-negative), yeast cells, and filamentous fungi studied ( unglycosylated MPAC at 200 M under identical conditions. Due to the particular mode of action, recently proposed for the proline-rich antimicrobial peptides and which proceeds through interaction with intracellular target molecules (28,29), we first examined the synergy between unglycosylated MPAC and a membrane-active antimicrobial peptide, Drosophila cecropin A (CecA). In the presence of CecA at a concentration twice below the MIC value, where the microorganisms are growing, a decrease in the MIC value of MPAC was observed against most of the Gram-negative bacteria tested (Table I).…”

The insect antimicrobial peptide, -pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK

“…The main findings from previous studies [4,20] of PYRDnaK interactions are summarized as follows: (i) L L -PYR inhibits the ATPase activity of DnaK; (ii) biotinylated-L L -PYR specifically binds to DnaK (590-615), which is a fragment containing the aD-aE helices; (iii) biotinylated-L L -PYR nonspecifically binds to both DnaK (397-439), which is a fragment of the substrate-binding domain, and to DnaK (596-637), which constitutes the aE helix and the 30-residue flexible Cterminus; and (iv) the N-terminal residues 1-9 of L L -PYR tightly bind to an 'allosteric ATPase site' on DnaK [20], which explains the ability of L L -PYR to inhibit DnaK-mediated ATP hydrolysis. Aspects of these earlier findings and their relation to our findings are discussed below.…”

“…According to recent reports, L L -PYR binds to wild-type DnaK at two sites, the conventional substrate-binding site and the aD and aE helices of the multi-helical lid [4,18]. In the experiments described below the binding of L L -PYR to the conventional substrate-binding site of DnaK was characterized.…”

“…Because the DnaK chaperone machine is absolutely essential to repair partially denatured proteins that occur as a consequence of stress, inhibiting DnaK would devastate a bacterial cell. There have been intriguing reports of novel classes of DnaK (Hsp70) [4] and DnaJ (Hsp40) [5] inhibitors. Such inhibitors, if they selectively bind to the prokaryotic but not to the eukaryotic chaperone, could be an important new class of antimicrobial agents.…”

Are Heat Shock Proteins Druggable Candidates?

“…For example, Hsp70s contain a fairly divergent C-terminal Peptide Binding Domain (PBD). Some proline-rich antibacterial peptides that target the PBD of DnaK (bacterial Hsp70) were reported not bind to human Hsp70 (Otvos et al, 2000). Hsp70s also possess sequence divergence in motifs that physically interact with their nucleotide exchange factors and for this reason, Hsp70s exhibit variable nucleotide exchange rates (Brehmer et al, 2001).…”