Melittin interaction with sulfated sugars and cell membranes

The presented work focused on an alternative mechanism of action of melittin on the cell membranes. The study using ITC reveals that melittin has a high affinity for several glycosaminoglycans (GAGs), i.e. heparan sulfate (HS), dermatan sulfate and heparin. The interaction between peptide and GAGs comprised both electrostatic and non-ionic components. Circular dichroism (CD) spectroscopy demonstrates that the binding of melittin to HS and other GAGs induces a conformational change to a predominantly α- helical structure. A model of the melittin-HS complex is presented. Furthermore the melittin binding was compared with that of magainin 2 and nisin Z to HS. Magainin 2 and nisin Z are two amphiphatic and antimicrobial peptides with similar lipid binding properties as melittin, but in opposite to melittin they do not cause lysis of the eukaryotic cells. ITC demonstrates that magainin 2 and nisin Z do not bind to HS. This result could indicate that the interaction with GAGs is unique property of melittin. In order to study in vivo involvement of GAGs in the binding of melittin to cell membranes, the cytotoxic effect of the peptide on the cells deficient in GAGs and the corresponding wild type cell line was investigated. The lactate dehydrogenase (LDH) release assay was employed. The differences in the toxic effect of the peptide on both cell lines were hoped to be much more pronounced, what would indicate that indeed GAGs are involved in the action mechanism of melittin on the cell membranes. The significant difference in the cytotoxic effect of melittin was observed only at one peptide concentration. However the surface of the eukaryotic cells is not only decorated with sulfated GAGs, but also with other negatively charged molecules and it cannot be excluded that they are also a potential target for melitin. Furthermore the interactions of retro-inverso dioleoylmelittin (riDOM) with HS were studied. RiDOM is a hybrid molecule, obtained by covalently coupling of retro-inverso analog of melittin to a lipid moiety, to form a stable and efficient gene transfer system, which shows no haemolytic activity. ITC demonstrates that riDOM has a high affinity to HS, and two other GAGs, namely dermatan sulfate and heparin. CD spectroscopy reveals no conformational changes of riDOM upon binding to HS. Dynamic light-scattering measurements showed formation of bigger aggregates/complexes when riDOM is titrated with HS. Both ionic and hydrophobic interactions contribute to the binding of riDOM to HS. Last part of the thesis described melittin-lipid membrane interaction using ITC. The binding equilibrium between melittin and electrically neutral large unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was studied. ITC demonstrates that melittin has a high affinity to neutral lipid membranes

Analysis of membrane interactions of antibiotic peptides using ITC and biosensor measurements

The interaction of the lantibiotic gallidermin and the glycopeptide antibiotic vancomycin with bacterial membranes was simulated using mass sensitive biosensors and isothermal titration calorimetry (ITC). Both peptides interfere with cell wall biosynthesis by targeting the cell wall precursor lipid II, but differ clearly in their antibiotic activity against individual bacterial strains. We determined the binding affinities of vancomycin and gallidermin to model membranes±lipid II in detail. Both peptides bind to DOPC/lipid II membranes with high affinity (K(D) 0.30 μM and 0.27 μM). Gallidermin displayed also strong affinity to pure DOPC membranes (0.53 μM) an effect that was supported by ITC measurements. A surface acoustic wave (SAW) sensor allowed measurements in the picomolar concentration range and revealed that gallidermin targets lipid II at an equimolar ratio and simultaneously inserts into the bilayer. These results indicate that gallidermin, in contrast to vancomycin, combines cell wall inhibition and interference with the bacterial membrane integrity for potent antimicrobial activity

Model membrane approaches to determine the role of calcium for the antimicrobial activity of friulimicin

Friulimicin is a cyclic lipopeptide antibiotic, currently in clinical development, that possesses excellent activity against Gram-positive bacteria, including multiresistant strains. A recent study on the mode of action of friulimicin reported on the interference with bacterial cell wall biosynthesis via a calcium-dependent complexing of the bactoprenol phosphate carrier C₅₅-P. The calcium dependency of this non-common targeted activity remains to be elucidated. In the present model membrane approach, the role of calcium for friulimicin targeting to C₅₅-P was investigated by biosensor-based detection of binding affinities. The findings were supplemented by atomic force microscopy (AFM) and circular dichroism (CD) spectroscopy. Comparing the calcium salt of friulimicin with the calcium-free peptide, calcium appeared to be essential for friulimicin interaction with DOPC model membranes. The binding affinity was even higher in the presence of 0.1 mol% C₅₅-P (0.21 μM vs. 1.22 μM), confirming the targeted mode of action. Binding experiments with supplemented calcium salts suggest (i) the phosphate group as the essential moiety of C₅₅-P, referring to a bridging function of calcium between the negatively charged friulimicin and C₅₅-P, and (ii) a structural effect of calcium shifting the peptide into a suitable binding conformation (CD spectra). AFM images confirmed that calcium has no, or only a minor, effect on the aggregate formation of friulimicin. These data shed new light on the mechanisms of antibacterial activity of friulimicin

riDOM, a Cell-Penetrating Peptide : Interaction with DNA and Heparan Sulfate

DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 10(7) M(-1) and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 10(6) M(-1) and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 10(6) M(-1). Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin