Abstract
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.
Similar content being viewed by others
Abbreviations
- AMP:
-
Antimicrobial peptide
References
Agerberth B, Lee JY, Bergman T et al (1991) Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. Eur J Biochem 202:849–854
Benincasa M, Scocchi M, Podda E et al (2004) Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates. Peptides 25:2055–2061
Bischofberger P, Han W, Feifel B, Schonfeld HJ, Christen P (2003) D-Peptides as inhibitors of the DnaK/DnaJ/GrpE chaperone system. J Biol Chem 278:19044–19047
Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250
Bulaj G, Kortemme T, Goldenberg DP (1998) Ionization-reactivity relationships for cysteine thiols in polypeptides. Biochemistry 37:8965–8972
Casteels P, Ampe C, Jacobs F, Vaeck M, Tempst P (1989) Apidaecins: antibacterial peptides from honeybees. EMBO J 8:2387–2391
Chan YR, Gallo RL (1998) PR-39, a syndecan-inducing antimicrobial peptide, binds and affects p130(Cas). J Biol Chem 273:28978–28985
Chesnokova LS, Slepenkov SV, Witt SN (2004) The insect antimicrobial peptide, l-pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK. FEBS Lett 565:65–69
Cho JH, Park CB, Yoon YG, Kim SC (1998) Lumbricin I, a novel proline-rich antimicrobial peptide from the earthworm: purification, cDNA cloning and molecular characterization. Biochim Biophys Acta 1408:67–76
Cudic M, Otvos L Jr (2002) Intracellular targets of antibacterial peptides. Curr Drug Targets 3:101–106
Destoumieux D, Bulet P, Loew D et al (1997) Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda). J Biol Chem 272:28398–28406
Feifel B, Sandmeier E, Schonfeld HJ, Christen P (1996) Potassium ions and the molecular-chaperone activity of DnaK. Eur J Biochem 237:318–321
Feifel B, Schonfeld HJ, Christen P (1998) D-peptide ligands for the co-chaperone DnaJ. J Biol Chem 273:11999–12002
Frank RW, Gennaro R, Schneider K, Przybylski M, Romeo D (1990) Amino acid sequences of two proline-rich bactenecins. Antimicrobial peptides of bovine neutrophils. J Biol Chem 265:18871–18874
Gaczynska M, Osmulski PA, Gao Y, Post MJ, Simons M (2003) Proline- and arginine-rich peptides constitute a novel class of allosteric inhibitors of proteasome activity. Biochemistry 42:8663–8670
Gallo RL, Ono M, Povsic T et al (1994) Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proc Natl Acad Sci USA 91:11035–11039
Gennaro R, Skerlavaj B, Romeo D (1989) Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils. Infect Immun 57:3142–3146
Gennaro R, Scocchi M, Merluzzi L, Zanetti M (1998) Biological characterization of a novel mammalian antimicrobial peptide. Biochim Biophys Acta 1425:361–368
Gennaro R, Zanetti M, Benincasa M, Podda E, Miani M (2002) Pro-rich antimicrobial peptides from animals: structure, biological functions and mechanism of action. Curr Pharm Des 8:763–778
Hale JD, Hancock RE (2007) Alternative mechanisms of action of cationic antimicrobial peptides on bacteria. Expert Rev Anti Infect Ther 5:951–959
Han W, Christen P (2003) Mechanism of the targeting action of DnaJ in the DnaK molecular chaperone system. J Biol Chem 278:19038–19043
Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381:571–579
Hartl FU, Martin J, Neupert W (1992) Protein folding in the cell: the role of molecular chaperones Hsp70 and Hsp60. Annu Rev Biophys Biomol Struct 21:293–322
Jenssen H, Hamill P, Hancock RE (2006) Peptide antimicrobial agents. Clin Microbiol Rev 19:491–511
Kay BK, Williamson MP, Sudol M (2000) The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J 14:231–241
Kragol G, Lovas S, Varadi G et al (2001) The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry 40:3016–3026
Kragol G, Hoffmann R, Chattergoon MA et al (2002) Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin. Eur J Biochem 269:4226–4237
Laufen T, Mayer MP, Beisel C et al (1999) Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci USA 96:5452–5457
Liebscher M, Roujeinikova A (2009) Allosteric coupling between the lid and interdomain linker in DnaK revealed by inhibitor binding studies. J Bacteriol 191:1456–1462
Liebscher M, Jahreis G, Lucke C et al (2007) Fatty acyl benzamido antibacterials based on inhibition of DnaK-catalyzed protein folding. J Biol Chem 282:4437–4446
Liu X (2002) Concentrations of the GroEL/GroES and the DnaK/DnaJ/GrpE molecular chaperones in Escherichia coli under normal and heat shock conditions. M.D. thesis. Universität Zürich, Zürich, Switzerland
Mattiuzzo M, Bandiera A, Gennaro R et al (2007) Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides. Mol Microbiol 66:151–163
Mogk A, Tomoyasu T, Goloubinoff P et al (1999) Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB. EMBO J 18:6934–6949
Osborn MJ, Munson R (1974) Separation of the inner (cytoplasmic) and outer membranes of gram-negative bacteria. Methods Enzymol 31:642–653
Otvos L Jr (2002) The short proline-rich antibacterial peptide family. Cell Mol Life Sci 59:1138–1150
Otvos L Jr, Rogers ME, Consolvo PJ et al (2000) Interaction between heat shock proteins and antimicrobial peptides. Biochemistry 39:14150–14159
Pierpaoli EV, Gisler SM, Christen P (1998) Sequence-specific rates of interaction of target peptides with the molecular chaperones DnaK and DnaJ. Biochemistry 37:16741–16748
Podda E, Benincasa M, Pacor S et al (2006) Dual mode of action of Bac7, a proline-rich antibacterial peptide. Biochim Biophys Acta 1760:1732–1740
Reddy KV, Yedery RD, Aranha C (2004) Antimicrobial peptides: premises and promises. Int J Antimicrob Agents 24:536–547
Rudiger S, Germeroth L, Schneider-Mergener J, Bukau B (1997) Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J 16:1501–1507
Rudiger S, Schneider-Mergener J, Bukau B (2001) Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone. EMBO J 20:1042–1050
Schmid D, Baici A, Gehring H, Christen P (1994) Kinetics of molecular chaperone action. Science 263:971–973
Schonfeld HJ, Schmidt D, Schroder H, Bukau B (1995a) The DnaK chaperone system of Escherichia coli: quaternary structures and interactions of the DnaK and GrpE components. J Biol Chem 270:2183–2189
Schonfeld HJ, Schmidt D, Zulauf M (1995b) Investigation of the molecular chaperone DnaJ by analytical ultracentrifugation. Prog Colloid Polym Sci 99:7–10
Shai Y (2002) Mode of action of membrane active antimicrobial peptides. Biopolymers 66:236–248
Shamova O, Brogden KA, Zhao C et al (1999) Purification and properties of proline-rich antimicrobial peptides from sheep and goat leukocytes. Infect Immun 67:4106–4111
Shi J, Ross CR, Leto TL, Blecha F (1996) PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox. Proc Natl Acad Sci USA 93:6014–6018
Stensvag K, Haug T, Sperstad SV et al (2008) Arasin 1, a proline-arginine-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. Dev Comp Immunol 32:275–285
Tomasinsig L, Zanetti M (2005) The cathelicidins: structure, function and evolution. Curr Protein Pept Sci 6:23–34
Tomoyasu T, Mogk A, Langen H, Goloubinoff P, Bukau B (2001) Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol. Mol Microbiol 40:397–413
Zhang L, Falla TJ (2006) Antimicrobial peptides: therapeutic potential. Expert Opin Pharmacother 7:653–663
Zhu X, Zhao X, Burkholder WF et al (1996) Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272:1606–1614
Acknowledgments
We thank Prof. Alessandro Tossi for critically reading the manuscript. This study was supported by grants from the Italian Ministry for University and Research (PRIN 2007) and from the Friuli Venezia Giulia Region (grant under the LR 26/2005, art. 23 for the R3A2 Network) to R. G., and from the Stiftung für Medizinische Forschung und Entwicklung, Zürich, to P. C.
Author information
Authors and Affiliations
Corresponding author
Additional information
Marco Scocchi and Christine Lüthy contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Scocchi, M., Lüthy, C., Decarli, P. et al. The Proline-rich Antibacterial Peptide Bac7 Binds to and Inhibits in vitro the Molecular Chaperone DnaK. Int J Pept Res Ther 15, 147–155 (2009). https://doi.org/10.1007/s10989-009-9182-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10989-009-9182-3