Proline-rich Antibacterial Peptide Research Articles

Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against Escherichia coli.

Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family. In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of E. coli and S. aureus as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated. The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against E. coli ATCC 25922 was not remarkably affected by the serum salts except for CaCl2 at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 μg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 μg/mL (8 × MICs) could effectively kill E. coli ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC25922. OaBac5mini is a stable and potent PrAMP that kills E. coli by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.

Directed chemical dimerisation enhances the antibacterial activity of the antimicrobial peptide MSI-78(4–20)

Antimicrobial resistance (AMR) is on the rise, leading to 700 000 deaths worldwide in 2020. Antimicrobial peptides (AMPs) are antibiotic agents that are active against multi-drug resistant pathogens and also have a reduced risk of AMR development. Previous studies have shown that dimerisation of the proline-rich antibacterial peptide (PrAMP) Chex1–Arg20 can enhance its antimicrobial activity while also reducing its toxicity. To determine if dimerisation via a simple disulfide bond can similarly improve other classes of AMPs, the α-helical cationic peptide MSI-78(4–20) was used as a model. The monomer alone, an S-carboxamidomethyl-capped N-terminal Cys–MSI-78(4–20) analogue and the disulfide-linked dimer were successfully synthesised and their antimicrobial activity and toxicity were determined. It was shown that dimerisation enhanced antimicrobial activity against the Gram-positive opportunistic pathogen Staphylococcus aureus ATCC 29213, the Gram-negative bacteria Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 47615. The peptides showed no significant haemolytic activity with red blood cells and only induced 50% lactate dehydrogenase (LDH) release in mammalian cells at the highest tested concentration, 15 µM. The MSI-78(4–20) dimer was less cytotoxic than the monomer and S-alkyl monomer. Together, the data support the strategy of AMP chemically directed dimerisation as a means of producing potentially more therapeutically useful antimicrobial agents.

Effects of Lipidation on a Proline-Rich Antibacterial Peptide.

The emergence of multidrug-resistant bacteria is a worldwide health problem. Antimicrobial peptides have been recognized as potential alternatives to conventional antibiotics, but still require optimization. The proline-rich antimicrobial peptide Bac7(1-16) is active against only a limited number of Gram-negative bacteria. It kills bacteria by inhibiting protein synthesis after its internalization, which is mainly supported by the bacterial transporter SbmA. In this study, we tested two different lipidated forms of Bac7(1-16) with the aim of extending its activity against those bacterial species that lack SbmA. We linked a C12-alkyl chain or an ultrashort cationic lipopeptide Lp-I to the C-terminus of Bac7(1-16). Both the lipidated Bac-C12 and Bac-Lp-I forms acquired activity at low micromolar MIC values against several Gram-positive and Gram-negative bacteria. Moreover, unlike Bac7(1-16), Bac-C12, and Bac-Lp-I did not select resistant mutants in E. coli after 14 times of exposure to sub-MIC concentrations of the respective peptide. We demonstrated that the extended spectrum of activity and absence of de novo resistance are likely related to the acquired capability of the peptides to permeabilize cell membranes. These results indicate that C-terminal lipidation of a short proline-rich peptide profoundly alters its function and mode of action and provides useful insights into the design of novel broad-spectrum antibacterial agents.

Charting the sequence-activity landscape of peptide inhibitors of translation termination

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

Sub-MIC effects of a proline-rich antibacterial peptide on clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii is one of the most important nosocomial pathogens, mainly due to its ability to accumulate antibiotic-resistances and to persist in the hospital environment - characteristics related to biofilm production. It is well-known that A. baumannii is inhibited by the proline-rich peptide Bac7(1-35), but its putative effects at sub-MICs were never considered. We examined the sub-MIC effect of Bac7(1-35) on the growth rate, resistance induction and some A. baumannii features linked to virulence. Growth kinetics in the presence of sub-MICs of Bac7(1-35) were evaluated spectrophotometrically. Peptide uptake was quantified by cytometric analysis. The ability of Bac7(1-35) to interfere with biofilm production was investigated by the crystal violet method and confocal microscopy. Bacterial motility was observed at the interphase between a layer of a semi-solid medium and the polystyrene bottom of a Petri dish. The induction of resistance was evaluated after serial passages with sub-MICs of the peptide. Although the MIC of Bac7(1-35) was between 2-4 µM for all tested strains, its effect on the growth rate at sub-MICs was strain-dependent and correlated with the amount of peptide internalized by each strain. Sub-MICs of Bac7(1-35) induced a strongly strain-dependent effect on biofilm formation and reduced motility in almost all strains, but interestingly the peptide did not induce resistance. Bac7(1-35) is internalized into A. baumannii and is able to inhibit biofilm formation and bacterial motility, without inducing resistance. This study stresses the importance of considering possible effects that antimicrobials could have at sub-MICs, mimicking a common condition during antibiotic treatment.

High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin

Drug resistance is a major problem in antibacterial chemotherapy. Apidaecins, which refer to a series of small, proline-rich antimicrobial peptides, are predominantly active against many drug-resistant bacteria. The apidaecins have special antibacterial mechanisms, and are non-toxic for human cells, a prerequisite for using them as novel antibiotic drugs. However, no efficient non-tagged apidaecin expression system has been reported, which is the limiting factor for their application. Here we successfully generated a Pichia pastoris transformant expressing and secreting apidaecin. However, expression was unstable and poor. Analysis of this revealed that the integration plasmid was frequently lost and that apidaecin expression resulted in cell death. Using N-methyl-N-nitro-N-nitroso-guanidine mutagenesis and selection, a mutant strain Apmu4 was derived, in which the rate of loss of the integration plasmid was much lower after induction, and which produced improved titres of apidaecin. Additionally, we discovered that using glucose as the sole carbon source to pre-culture the strain before induction could greatly enhance apidaecin production. A pilot-scale 10 L fermentation yielded 418 mg/L of recombinant apidaecin, which represents the highest reported yield of apidaecin. Consequently, this study reports the first super heterologous expression and secretion of apidaecin in yeast.

PEGylation of the peptide Bac7(1–35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity

The proline-rich antibacterial peptide Bac7(1–35) protects mice against Salmonella typhimurium infection, despite its rapid clearance. To overcome this problem the peptide was linked to a polyethylene glycol (PEG) molecule either via a cleavable ester bond or via a non-hydrolysable amide bond. Both the PEGylated conjugates retained most of the in vitro activity against S. typhimurium. In addition, the ester bond was cleaved in human serum or plasma, releasing a carboxymethyl derivative of Bac7(1–35) which accounts for a higher activity of this peptide with relative to the other, non-hydrolysable form. Both PEGylated peptides maintained the capacity of the unconjugated form to kill bacteria without permeabilizing the bacterial membranes, by penetrating into cells. They exploited the same transporter as unmodified Bac7(1–35), suggesting it has the capacity to internalize quite sizeable cargo if this is linked to Bac7 fragment. PEGylation allows the peptide to have a wide distribution in mice, and a slow renal clearance, indicating that this strategy would improve the bioavailability of Bac7, and in principle of other antimicrobial peptides. This can be an equally important issue to reducing cytotoxicity for therapeutic use of these antibacterials.

The Designer Proline-rich Antibacterial Peptide A3-APO Prevents Bacillus anthracis Mortality by Deactivating Bacterial Toxins

Proline-rich antibacterial peptides protect experimental animals from bacterial challenge even if they are unable to kill the microorganisms in vitro. Their major in vivo modes of action are inhibition of bacterial protein folding and immunostimulation. Here we investigated whether the proline-rich antibacterial peptide dimer A3-APO was able to inhibit Bacillus cereus enterotoxin production in vitro and restrict the proliferation of lethal toxin-induced Bacillus anthracis replication in mouse macrophages. After 24 h incubation, peptide A3-APO and its single chain metabolite reduced the amount of properly folded B. cereus diarrhoeal enterotoxin production in a concentration-dependent manner leading to only 10-25% of the original amount of toxin detectable by a conformation-sensitive immunoassay. Likewise, after 4 h incubation, A3-APO restricted the proliferation of B. anthracis in infected macrophages by 40-45% compared to untreated cells both intracellularly and in the extracellular cell culture milieu. Although the peptide had a minimal inhibitory concentration of >512 mg/L against B. anthracis in vitro, in systemic mouse challenge models it improved survival by 20- 37%, exhibiting statistically significant cumulative efficacy when administered at 3x5 mg/kg intraperitoneally or intramuscularly. We hypothesize that the activity in isolated murine macrophages and in vivo is due to deactivation of bacterial toxins. Bacterial protein folding inhibition in synergy with other types of antimicrobial modes offers a remarkable novel strategy in combating resistant or life-threatening infections.

The designer proline-rich antibacterial peptide A3-APO is effective against systemic Escherichia coli infections in different mouse models

Antimicrobial peptides are considered to be viable alternatives to conventional antibiotics. However, they rarely show systemic efficacy in animal models when added at non-toxic doses. The dimer A3-APO was designed to attack both the bacterial membrane and the Enterobacteriaceae-specific domain of the heat shock protein DnaK in order to reduce toxicity whilst maintaining activity. The peptide exhibited a minimal inhibitory concentration (MIC) range of 2–128 mg/L against 28 clinical Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium strains, with a median MIC of 30 mg/L. At this concentration, A3-APO was bactericidal to E. coli 5770, a fluoroquinolone-resistant extended-spectrum β-lactamase-producing strain. The No Observed Adverse Effect Limit (NOAEL) at repeated intraperitoneal peptide administration was 20 mg/kg. When administered at this dose three times starting immediately after E. coli Neumann infection, A3-APO cured 100% of mice in a standard bacteraemia model used by the pharmaceutical industry. In a more stringent assay, when treatment started after E. coli 5770 bacteraemia had already been established, three doses of 10 mg/kg A3-APO prolonged early survival at a rate similar to that of imipenem and reduced the bacterial counts to base level. When the second assay was repeated in kidney clearance conditions resembling those in humans, 10 mg/kg A3-APO was as efficacious as imipenem in the long-term. The increased in vivo efficacy compared with the in vitro bactericidal figures can potentially be explained by the generally observable immunostimulatory properties of antimicrobial peptides. Peptide A3-APO shows promising features as a member in our antibiotic arsenal against multidrug-resistant bacterial pathogens.

The Antibacterial Effect of a Proline-Rich Antibacterial Peptide A3-APO

Antimicrobial resistance is an emerging worldwide concern in light of the widespread antimicrobial drug use in humans, livestock and companion animals. The treatment of life-threatening infections is especially problematic because clinical strains rapidly acquire multiple-drug resistance. Antimicrobial peptides have long been considered to be viable alternatives to small molecule antibiotics. However, the peptides' parenteral use is frequently hampered by inadequate safety margins and rapid renal clearance leaving them suitable only for topical applications. The proline-rich peptide A3-APO represents a family of a new class of synthetic dimers that kill bacteria by a dual mode of action and carry domains for interaction with both the bacterial membrane and an intracellular target. From a series of designer antibacterial peptides, A3-APO emerged as a viable preclinical candidate by virtue of its superior ability to disintegrate the bacterial membrane, inhibit the 70-kDa heat shock protein DnaK alone or in synergy with small molecule antibiotics, lack of eukaryotic toxicity and withstand proteolytic degradation in body fluids. As many other proline-rich peptides, A3-APO binds to the C-terminal helical lid of bacterial DnaK and inhibits chaperone-assisted protein folding in bacteria but not in mammalian Hsp70. In this review, the structure, pharmacokinetic properties, antimicrobial spectrum of peptide A3-APO and its in vivo metabolite are summarized and the in vitro and in vivo antimicrobial effects (antimicrobial susceptibilities, postantibiotic effects, resistance induction) are discussed in detail.

The Proline-rich Antibacterial Peptide Bac7 Binds to and Inhibits in vitro the Molecular Chaperone DnaK

Bac7, a cathelicidin peptide of the proline-rich group, inactivates bacteria in a stereospecific manner by entering target cells without any apparent membrane damage and by binding to as yet unknown intracellular targets. The present study was aimed at detecting these putative intracellular interactors, which might mediate the antibacterial action of this peptide. By using affinity resins functionalized with the N-terminal 1-35 fragment of Bac7, a single protein was specifically retained with high affinity from Escherichia coli cytoplasmic protein lysates. This ligand was identified as the heat shock protein DnaK, the Hsp70 homolog in E. coli. The interaction between the peptide and the chaperone is stereospecific, given that a resin prepared with the all-d enantiomer failed to retain the protein. In vitro, Bac7(1-35) formed a complex with DnaK with an affinity comparable to that of other known high-affinity peptide ligands. In addition, at 10-100 μM concentration, the peptide inhibited the protein refolding activity of the complete DnaK/DnaJ/GrpE/ATP molecular chaperone system in a dose-dependent manner. Despite these results, the in vitro sensitivity to the peptide, under growth permitting conditions, of DnaK-deficient E. coli strains was not significantly affected compared to the wild-type strain. This suggests that, apart from DnaK, other vital targets for the proline-rich AMPs are present in susceptible bacteria.

Scope and limitations of the designer proline-rich antibacterial peptide dimer, A3-APO, alone or in synergy with conventional antibiotics

The proline-rich antimicrobial peptide dimer, A3-APO, was designed based on a statistical analysis of native antibacterial peptide and protein sequences. Analysis of a series of structural analogs failed to identify any single or multiple amino acid modification or architectural changes that would significantly improve its potential as a clinical therapeutic. However, a single chain Chex1-Arg20 version, a natural in vivo metabolite, showed a 2 to 8-fold increase in activity against test Enterobacteriaceae strains. In addition to bacterial species close to Escherichia coli in phylogeny, A3-APO analogs were able to effectively kill Pseudomonas aeruginosa and Staphylococcus saprophyticus. Antibacterial efficacy analysis together with biochemical experiments provided further evidence for a multiple mode of action of A3-APO that includes binding and inhibition of the bacterial heat shock protein DnaK. Through inactivating of resistance enzymes, A3-APO was able to recover the lost activity of conventional antibiotics including chloramphenicol, β-lactams, sulfonamides or trimethoprim against multidrug resistant strains with partial or full synergy. However, the synergy appeared to be individual strain and small molecule drug combination-dependent.

Designer Antibacterial Peptides Kill Fluoroquinolone-Resistant Clinical Isolates

A significant number of Escherichia coli and Klebsiella pneumoniae bacterial strains in urinary tract infections are resistant to fluoroquinolones. Peptide antibiotics are viable alternatives although these are usually either toxic or insufficiently active. By applying multiple alignment and sequence optimization steps, we designed multifunctional proline-rich antibacterial peptides that maintained their DnaK-binding ability in bacteria and low toxicity in eukaryotes, but entered bacterial cells much more avidly than earlier peptide derivatives. The resulting chimeric and statistical analogues exhibited 8-32 microg/mL minimal inhibitory concentration efficacies in Muller-Hinton broth against a series of clinical pathogens. Significantly, the best peptide, compound 5, A3-APO, retained full antibacterial activity in the presence of mouse serum. Across a set of eight fluoroquinolone-resistant clinical isolates, peptide 5 was 4 times more potent than ciprofloxacin. On the basis of the in vitro efficacy, toxicity, and pharmacokinetics data, we estimate that peptide 5 will be suitable for treating infections in the 3-5 mg/kg dose range.

A novel lebocin-like gene from eri-silkworm, Samia cynthia ricini, that does not encode the antibacterial peptide lebocin

A cDNA clone with homology to lebocin gene was isolated from fat body of immunized Samia cynthia ricini larvae. The cDNA has an open reading frame encoding 162 amino acid residues. The deduced amino acid sequence shows significant homology to lebocin precursor proteins from Bombyx mori and Trichoplusia ni only in the “prosegment” region, but no homology to mature lebocin, a proline-rich antibacterial peptide, indicating the protein is not a precursor for lebocin antibacterial peptide. Northern analysis indicates that transcript of the lebocin-like gene is not detected in any tissues of naive larvae, but induced mainly in fat body after injection of the larvae with bacterial cells or cell wall components, such as peptidoglycan.

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

In Drosophila melanogaster, seven distinct families of antimicrobial peptides with different structures and specificities are synthesized by the fat body and released into the hemolymph during the immune response. Using microscale high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Edman degradation, we have isolated and characterized from immune-challenged Drosophila two novel induced molecules, under the control of the Imd pathway, that correspond to post-translationally modified antimicrobial peptides or peptide fragments. The first molecule is a doubly glycosylated form of drosocin, an O-glycosylated peptide that kills Gram-negative organisms. The second molecule represents a truncated form of the pro-domain of the Drosophila attacin C carrying two post-translational modifications and has significant structural similarities to proline-rich antibacterial peptides including drosocin. We have synthesized this peptide and found that it is active against Gram-negative bacteria. Furthermore, this activity is potentiated when the peptide is used in combination with the Drosophila antimicrobial peptide cecropin A. The synergistic action observed between these two molecules suggests that the truncated post-translationally modified pro-domain of attacin C by itself may play an important role in the antimicrobial defense of Drosophila.

Walking the fine line between intracellular and membrane activities of antibacterial peptides

Analogs of pyrrhocoricin, a proline-rich antibacterial peptide with a potential therapeutic use, show multiple actions on bacterial cells. We used a dual-fluorochrome membrane viability assay to provide evidence that the lead drug candidate, Pip-pyrr-MeArg dimer derivative, kills bacteria better than the native peptide due to an improved activity on bacterial membranes. This assay was also instrumental in documenting that activity on bacterial membranes and toxicity to human cells can be correlated, and the predominant mode of action can be changed from intracellular DnaK inhibition to membrane disintegration. Similar analyses with an alanine-scan on pyrrhocoricin identified Lys3 as a crucial player to interaction with bacterial membranes, three prolines in mid-chain position as being responsible for maintaining structural integrity and Asp2, Tyr6, Leu7, and Arg9 as putative contact points to the D-E helix of the bacterial target protein DnaK.

Neutralization of endotoxin in vitro and in vivo by Bac7(1-35), a proline-rich antibacterial peptide.

Lipopolysaccharides (LPS), or endotoxins, are structural components of gram-negative bacteria implicated in the pathogenesis of septic shock. In this study the antiendotoxin activity of Bac7(1-35), a synthetic peptide based on the sequence of a proline-rich antibacterial peptide from bovine neutrophils, was investigated in vitro and in an experimental rat model of gram-negative septic shock. The ability of Bac7(1-35) to bind LPS from Escherichia coli O111:B4 was determined using a sensitive Limulus chromogenic assay. In the in vivo study, adult male Wistar rats were given an intraperitoneal injection of 1 x 10(9) colony-forming units of E. coli ATCC 25922. All animals were randomized to receive intraperitoneally 1 mg/kg Bac7(1-35), or isotonic sodium chloride solution (control group C1), 60 mg/kg of piperacillin and 1 mg/kg polymyxin B, 1 mg/kg of polymyxin B plus 60 mg/kg of piperacillin, and 1 mg/kg of Bac7(1-35) plus 60 mg/kg of piperacillin. Each group included 15 animals. Bac7(1-35) was found to completely inhibit the LPS procoagulant activity at approximately 10 microM peptide concentration, as determined by in vitro LAL chromogenic assay. Treatment with Bac7(1-35) resulted in significant decrease in plasma endotoxin levels and lethality rates compared with saline injected control animals. No statistically significant differences were noted between Bac7(1-35) and polymyxin B in reducing all variables measured. These results provide evidence for the ability of Bac7(1-35) to effectively bind LPS and protect animals from lethal effects of this molecule, and point to its potential use for the treatment of endotoxin-induced septic shock.

Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin.

Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure-antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2-10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin.

The short proline-rich antibacterial peptide family.

From the many peptide families that are induced upon bacterial infection and can be isolated from all classes of animals, the short, proline-rich antibacterial peptides enjoy particular interest. These molecules were shown to inactivate an intracellular biopolymer in bacteria without destroying or remaining attached to the bacterial cell membrane, and as such emerged as viable candidates for the treatment of mammalian infections. These peptides were originally isolated from insects, they kill mostly gram-negative bacteria with high efficiency and they show structural similarities with longer insect- and mammal-derived antimicrobial peptides. However, while the distant relatives appear to carry multiple functional domains, apidaecin, drosocin, formaecin and pyrrhocoricin consist of only minimal determinants needed to penetrate across the cell membrane and bind to the target biopolymer. These peptides appear to inhibit metabolic processes, such as protein synthesis or chaperone-assisted protein folding. Pyrrhocoricin derivatives protect mice from experimental infections in vivo, suggesting the utility of modified analogs in the clinical setting. Sequence variations of the target protein at the peptide-binding site may allow the development of new peptide variants that kill currently unresponsive strains or species.

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding.

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.