Proc Natl Acad Sci U S A 111(14): E1409-E1418

PMC: PMC3986158

PMID: 24706874

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

Maya Penkova+9 authors

Supplementary Material

Supporting Information:

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^{Biology of Microorganisms,}

^{Bioinorganic Chemistry, and}

^{Plant Physiology,}

^{Immune Proteomics, Medical Proteome Center, and}

^{Institute of Physiological Chemistry, Ruhr University Bochum, 44801 Bochum, Germany;}

^{Institute for Medical Microbiology, Immunology, and Parasitology, Pharmaceutical Microbiology Section, University of Bonn, 53113 Bonn, Germany;}

^{Department of Microbial Physiology and Molecular Biology, Ernst Moritz Arndt University, 17489 Greifswald, Germany;}

^{Biophysics Division, Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria;}

^{Institute for Pharmaceutical Biology and Biotechnology, Heinrich Heine University, 40225 Düsseldorf, Germany;}

^{Department of Pharmaceutical Chemistry, University of Innsbruck, 6020 Innsbruck, Austria;}

^{Department of Chemistry, University of British Columbia, Vancouver, BC, Canada, V6T 1Z1; and}

^{Department of Biology, University of Marburg, 35037 Marburg, Germany}

^{To whom correspondence should be addressed. E-mail:}ed.bur@wodnab.ailuj.

Edited by Michael Zasloff, Georgetown University Medical Center, Washington, DC, and accepted by the Editorial Board February 27, 2014 (received for review October 22, 2013)

Author contributions: M.W., H.-G.S., and J.E.B. designed research; M.W., A.I.C., A.O., D.Z., C.M., C.S., and R.G. performed research; H.B.A., M.P., U.K., N.M.-N., S.K.S., E.B., D.B., and H.B.-O. contributed new reagents/analytic tools; M.W., A.I.C., A.O., D.Z., C.M., C.S., R.G., R.E., H.B.-O., and J.E.B. analyzed data; and M.W., H.B.-O., H.-G.S., and J.E.B. wrote the paper.

Edited by Michael Zasloff, Georgetown University Medical Center, Washington, DC, and accepted by the Editorial Board February 27, 2014 (received for review October 22, 2013)

Significance

Multidrug-resistant bacteria present an acute problem to medicine, generating interest in novel antimicrobial strategies. Antimicrobial peptides currently are being investigated, both as antibiotics and as immunomodulatory agents. Many antimicrobial peptides interact with the bacterial membrane, a previously underexplored antibiotic target. We present a system-based study of the mode of action of small cationic peptides and the mechanisms that bacteria use to defend against them. We show that peptide integration into the membrane causes delocalization of essential peripheral membrane proteins. This delocalization impacts on two cellular processes, namely respiration and cell-wall biosynthesis. We describe a bacterial survival strategy in which mechanosensitive channels in the bacterial membrane establish osmoprotection against membrane-targeting bacteriolytic peptides. Understanding the peptides' mode of action and bacterial survival strategies opens up new avenues for devising peptide-based antibacterial strategies.

Keywords: mechanism of action, respiratory chain, hypoosmotic stress response, metallocenes

Significance

Abstract

Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH₂. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions.

Abstract

During the past two decades, bacterial antibiotic resistance has grown into a major threat to public health, restoring infectious diseases to the list of leading causes of death worldwide (www.who.int/healthinfo/statistics/bodgbddeathdalyestimates.xls). As a reaction to this health crisis, several efforts currently are underway to discover and develop new natural and synthetic antibiotic compounds. One promising antibiotic class is antimicrobial peptides, which occur naturally as part of host defense systems in all domains of life (1 –4). Antimicrobial peptides range in length from four to more than 100 amino acids and fall into a number of different structural classes, including α-helical amphiphiles, lipopeptides, glycopeptides, lantibiotics, and short cationic peptides.

The short cationic peptides offer a potent alternative to longer natural antimicrobial peptides. The latter can be difficult to isolate and are complicated to synthesize chemically, but short cationic peptides are generated readily by solid-phase peptide synthesis and are easily accessible for chemical derivatization (5 –8). They are characterized by positively charged and by hydrophobic amino acids (9, 10). Previous mechanistic studies in vitro have examined the interactions of the peptides with membranes or membrane extracts (11 –15), but their effects on bacterial physiology have been largely underexplored.

A more complete understanding of how these short cationic antimicrobial peptides bring about bacterial cell death is needed to further their optimization and development for practical applications. To achieve this understanding, we studied the synthetic hexapeptide RWRWRW-NH₂ (referred to hereafter as “MP196”) (see SI Appendix, Fig. S1 for structure) as a model representing the minimal pharmacophore of positively charged and hydrophobic amino acids (16, 17). It is effective against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus strains, is moderately effective against Gram-negative bacteria, has low toxicity against human cell lines, and displays low hemolytic activity (18). MP196 therefore is a promising lead structure for derivatization and already has yielded peptides with improved activities and altered pharmacological properties (19). For example, in a recent systematic l-to-d exchange scan peptides with significantly reduced hemolytic activity could be identified (20).

Proteomic profiling of the Gram-positive bacterium Bacillus subtilis exposed to MP196 provided a starting point for mechanistic studies. It identified two major areas for analysis: membrane and cell-wall integrity and energy metabolism. The proteome analysis prompted further investigation into the deregulation of amino acid biosynthesis, which revealed that B. subtilis counteracts the attack on the cell envelope by triggering an osmoprotective release of glutamate.

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Acknowledgments

We thank Michaele Josten, Petra Düchting, Monika Bürger and Beate Menzel, and Stephanie Tautges, Kathrin Barlog, and Jale Stoutjesdijk for excellent technical assistance; Christoph H. R. Senges for help in preparing B. subtilis samples; Sina Langklotz and Dirk Albrecht for assistance with mass spectrometry; Helmut Meyer for providing amino acid analysis technology; Klaus Funke and Ellen Kloosterboer for providing the cytochrome c antibody; Tanneke de Blaauwen and Franz Naberhaus for providing the MurG antibody; and AiCuris, GmbH for providing the B. subtilis reporter strains. This manuscript was written in part at the 2013 Scientific Writing workshop of the Ruhr University Bochum (RUB) Research School; we thank Lars Leichert and Richard Gallagher for critically reading the manuscript and for many helpful suggestions. This work was supported by a grant from the German federal state of North Rhine-Westphalia and the European Union (European Regional Development Fund “Investing in your future”) (to N.M.-N., H.B.-O., H.-G.S., and J.E.B.) and by the RUB Research Department Interfacial Systems Chemistry (N.M.-N. and J.E.B.). C.M. was supported by the Protein Unit for Research in Europe, a project of North Rhine-Westphalia.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. M.Z. is a guest editor invited by the Editorial Board.

Data deposition: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [Vizcaino JA, et al. (2013) The Proteomics Identifications (PRIDE) database and associated tools: Status in 2013. Nucleic Acids Res 41(D1):D1063–D1069]. The dataset ID is PXD000181.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319900111/-/DCSupplemental.

Footnotes

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