Investigation of the Mechanisms of Antimicrobial Peptides Interacting with Membranes by Fluorescence Correlation Spectroscopy

The interaction of four antimicrobial peptides, including magainin 2, melittin, polymyxin B and an artificial peptide V4, with phospholipid membranes was investigated by fluorescence correlation spectroscopy (FCS) and confocal imaging. Fluorophore entrapping and labeled vesicles were used to quantitatively determine the extent of antimicrobial peptides interacting with membrane mimics and unravel their different mechanisms of action. It is shown that at high pep- tide/lipid ratio, magainin 2 and melittin form pores to induce comparable level of membrane permeation while polymyxin B and V4 disrupt the membrane. Considering its low solubility, V4 is much more active than others. Due to the hydro- phobic interactions, melittin, polymyxin B and V4 all promote aggregation when they perform their function. These ex- periments demonstrate the feasibility of mechanistic studies by an FCS based approach.

[1]  A. Ouellette,et al.  Combination of ellipsometry, laser scanning microscopy and Z‐scan fluorescence correlation spectroscopy elucidating interaction of cryptdin‐4 with supported phospholipid bilayers , 2008, Journal of peptide science : an official publication of the European Peptide Society.

[2]  G. Forrest,et al.  Polymyxin antibiotics for gram-negative infections. , 2007, American journal of health-system pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists.

[3]  R. Hancock,et al.  Cationic host defence peptides: Innate immune regulatory peptides as a novel approach for treating infections , 2007, Cellular and Molecular Life Sciences.

[4]  R. Hancock,et al.  Cationic host defense (antimicrobial) peptides. , 2006, Current opinion in immunology.

[5]  J. L. Ding,et al.  Determination of critical micelle concentrations and aggregation numbers by fluorescence correlation spectroscopy: aggregation of a lipopolysaccharide. , 2006, Analytica chimica acta.

[6]  A. Pramanik,et al.  Membrane perturbation effects of peptides derived from the N-termini of unprocessed prion proteins. , 2005, Biochimica et biophysica acta.

[7]  J. L. Ding,et al.  Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: an amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity. , 2005, Biochimica et biophysica acta.

[8]  U. Seydel,et al.  Probing the properties of lipopolysaccharide monolayers and their interaction with the antimicrobial peptide polymyxin B by atomic force microscopy. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[9]  Rudolf Rigler,et al.  Fluorescence correlation spectroscopy of molecular motions and kinetics. , 2005, Advanced drug delivery reviews.

[10]  Petra Schwille,et al.  Single-molecule spectroscopic methods. , 2004, Current opinion in structural biology.

[11]  B. Ho,et al.  De Novo Design of Potent Antimicrobial Peptides , 2004, Antimicrobial Agents and Chemotherapy.

[12]  S. McLaughlin,et al.  Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles. , 2004, Biophysical journal.

[13]  P. Schwille,et al.  Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience , 2003, Current Opinion in Neurobiology.

[14]  T. Ganz.  Defensins: antimicrobial peptides of innate immunity , 2003, Nature Reviews Immunology.

[15]  Petra Schwille,et al.  Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy. , 2003, Methods.

[16]  M. Zasloff.  Antimicrobial peptides of multicellular organisms , 2002, Nature.

[17]  Watt W Webb,et al.  Biological and chemical applications of fluorescence correlation spectroscopy: a review. , 2002, Biochemistry.

[18]  O. Krichevsky,et al.  Fluorescence correlation spectroscopy: the technique and its applications , 2002 .

[19]  Alessandro Tossi,et al.  Amphipathic α helical antimicrobial peptides. , 2001 .

[20]  L. Yang,et al.  Barrel-stave model or toroidal model? A case study on melittin pores. , 2001, Biophysical journal.

[21]  R. Rigler,et al.  The standard deviation in fluorescence correlation spectroscopy. , 2001, Biophysical journal.

[22]  K. Hahm,et al.  Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1-8)-magainin 2(1-12) and its analogues, on their antibiotic activities and structures. , 2000, Biochemistry.

[23]  Apostolov,et al.  Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles , 2000, Biophysical chemistry.

[24]  S. Blondelle,et al.  Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity. , 1999, Biochimica et biophysica acta.

[25]  Y. Shai,et al.  Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. , 1999, Biochimica et biophysica acta.

[26]  N. Surolia,et al.  Surface Plasmon Resonance Studies Resolve the Enigmatic Endotoxin Neutralizing Activity of Polymyxin B* , 1999, The Journal of Biological Chemistry.

[27]  R. Rigler,et al.  Resolution of fluorescence correlation measurements. , 1999, Biophysical journal.

[28]  A. Surolia,et al.  Kinetics of the interaction of endotoxin with polymyxin B and its analogs: a surface plasmon resonance analysis , 1999, FEBS letters.

[29]  K. Matsuzaki,et al.  Magainins as paradigm for the mode of action of pore forming polypeptides. , 1998, Biochimica et biophysica acta.

[30]  R. Rigler,et al.  Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces. , 1998, Cellular and molecular biology.

[31]  M. Wenk,et al.  Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation. , 1998, Biochemistry.

[32]  G. Schwarz,et al.  Quantitative studies on the melittin-induced leakage mechanism of lipid vesicles. , 1998, Biochemistry.

[33]  W. Webb,et al.  Fluorescence correlation spectroscopy: diagnostics for sparse molecules. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[34]  K. Miyajima,et al.  Pore formation and translocation of melittin. , 1997, Biophysical journal.

[35]  R. Hancock,et al.  Peptide antibiotics , 1997, The Lancet.

[36]  Peet Kask,et al.  Statistical accuracy in fluorescence fluctuation experiments , 1997, European Biophysics Journal.

[37]  S J Ludtke,et al.  Membrane pores induced by magainin. , 1996, Biochemistry.

[38]  N. Fujii,et al.  An antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation. , 1996, Biochemistry.

[39]  R. Hancock,et al.  Mode of Action of the Antimicrobial Peptide Indolicidin* , 1996, The Journal of Biological Chemistry.

[40]  London Wc,et al.  De Novo Antimicrobial Peptides with Low Mammalian Cell Toxicity , 1996 .

[41]  M. Lafleur,et al.  Study of vesicle leakage induced by melittin. , 1995, Biochimica et biophysica acta.

[42]  R. Hancock,et al.  Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification , 1994, Antimicrobial Agents and Chemotherapy.

[43]  S. Ludtke,et al.  Cooperative membrane insertion of magainin correlated with its cytolytic activity. , 1994, Biochimica et biophysica acta.

[44]  Richard G. Sleight,et al.  Fluorescence assay for phospholipid membrane asymmetry. , 1991, Biochemistry.

[45]  J. Blazyk,et al.  Interactions between the antimicrobial peptide, magainin 2, and Salmonella typhimurium lipopolysaccharides , 1991, FEBS letters.

[46]  J. Blazyk,et al.  Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure. , 1991, Biochemistry.

[47]  M. Zasloff,et al.  Magainins: a new family of membrane-active host defense peptides. , 1990, Biochemical pharmacology.

[48]  M. Zasloff,et al.  Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[49]  F. Jähnig,et al.  The structure of melittin in membranes. , 1986, Biophysical journal.

[50]  D. Morrison,et al.  Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. , 1976, Immunochemistry.

[51]  S. Aragon,et al.  Fluorescence correlation spectroscopy as a probe of molecular dynamics , 1976 .

[52]  Måns Ehrenberg,et al.  Rotational brownian motion and fluorescence intensify fluctuations , 1974 .

[53]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[54]  E Habermann,et al.  Bee and wasp venoms. , 1972, Science.

[55]  Received , 1868, Buffalo medical and surgical journal.

[56]  K. Berland,et al.  Fluorescence correlation spectroscopy: a new tool for quantification of molecular interactions. , 2004, Methods in molecular biology.

[57]  Keith M. Berland,et al.  Fluorescence Correlation Spectroscopy , 2004 .

[58]  J. Lakowicz.  Topics in fluorescence spectroscopy , 2002 .

[59]  R. Rigler,et al.  Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy. , 2000, Chemistry and physics of lipids.

[60]  Alessandro Tossi,et al.  Amphipathic, α‐helical antimicrobial peptides , 2000 .

[61]  Y. Shai,et al.  Mode of action of linear amphipathic α-helical antimicrobial peptides , 1998 .

[62]  S. Bishop,et al.  De novo antimicrobial peptides with low mammalian cell toxicity. , 1996, Journal of medicinal chemistry.