Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides

Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure–activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics.     

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa).The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition.We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.     

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.     

Membrane and lipopolysaccharide interactions of C-terminal peptides from S1 peptidases

The mechanisms underlying antimicrobial and anti-endotoxic effects were investigated for a series of structurally related peptides derived from the C-terminal region of S1 peptidases. For this purpose, results on bacterial killing were compared to those on peptide-induced liposome leakage, and to ellipsometry and dual polarization interferometry results on peptide binding to, and disordering of, supported lipid bilayers. Furthermore, the ability of these peptides to block endotoxic effects caused by bacterial lipopolysaccharide (LPS), monitored through NO production in macrophages, was compared to the binding of these peptides to LPS, and to secondary structure formation in the peptide/LPS complex. Bacteria killing, occurring through peptide-induced membrane lysis, was found to correlate with liposome rupture, and with the extent of peptide binding to the lipid membrane, no adsorption threshold for peptide insertion being observed. Membrane and LPS binding was found to depend on peptide net charge, illustrated by LPS binding increasing with increasing peptide charge, and peptides with net negative charge being unable to lyse membranes, kill bacteria, and block LPS-induced endotoxic effect. These effects were, however, also influenced by peptide hydrophobicity. LPS binding was furthermore demonstrated to be necessary, but not sufficient, for anti-endotoxic effect of these peptides. Circular dichroism spectroscopy showed that pronounced helix formation occurs in peptide/LPS complexes for all peptides displaying anti-endotoxic effect, hence potentially linked to this functionality. Similarly, ordered secondary structure formation was correlated to membrane binding, lysis, and antimicrobial activity of these peptides. Finally, preferential binding of these peptides to LPS over the lipid membrane was demonstrated.     

Lipopolysaccharides in bacterial membranes act like cholesterol in eukaryotic plasma membranes in providing protection against melittin-induced bilayer lysis

Melittin is a small, cationic peptide that, like many other antimicrobial peptides, lyses cell membranes by acting on their lipid bilayers. However, the sensitivity to antimicrobial peptides varies among cell types. We have performed direct binding and vesicle leakage experiments to determine the sensitivity to melittin of bilayers composed of various physiologically relevant lipids, in particular, key components of eukaryotic membranes (cholesterol) and bacterial outer membranes (lipopolysaccharide or LPS). Melittin binds to bilayers composed of both zwitterionic and negatively charged phospholipids, as well as to the highly charged LPS bilayers. The magnitude of the free energy of binding (deltaG degrees ) increases with increasing bilayer charge density; deltaG degrees = -7.6 kcal/mol for phosphatidylcholine (PC) bilayers and -8.9 to -11.0 kcal/mol for negatively charged bilayers containing phosphatidylserine (PS), phospholipids with covalently attached polyethylene glycol (PEG-lipids), or LPS. Comparisons of these data show that binding is not markedly affected by the steric barrier produced by the PEG in PEG-lipids or by the polysaccharide core of LPS. The addition of equimolar cholesterol to PC bilayers reduces the level of binding (deltaG degrees = -6.4 kcal/mol) and reduces the extent of melittin-induced leakage by 20-fold. LPS and 1:1 PC/cholesterol bilayers have similar high resistance to melittin-induced leakage, indicating that cholesterol in eukaryotic plasma membranes and LPS in Gram-negative bacteria provide strong protection against the lytic effects of melittin. We argue that this resistance is due at least in part to the similar tight packing of the lipid acyl chains in PC/cholesterol and LPS bilayers. The addition of bacterial phospholipids to LPS bilayers increases their sensitivity to melittin, helping to explain the higher sensitivity of deep rough bacteria compared to smooth phenotypes.     

Structural insights on the selective interaction of the histidine-rich piscidin antimicrobial peptide Of-Pis1 with membranes

Of-Pis1 is a potent piscidin antimicrobial peptide (AMP), recently isolated from rock bream (Oplegnathus fasciatus). This rich in histidines and glycines 24-amino acid peptide displays high and broad antimicrobial activity and no significant hemolytic toxicity against human erythrocytes, suggesting low toxicity. To better understand the mechanism of action of Of-Pis1 and its potential selectivity, using NMR and CD spectroscopies, we studied the interaction with eukaryotic and procaryotic membranes and membrane models. Anionic sodium dodecyl sulfate (SDS) and lipopolysaccharide (LPS) micelles were used to mimic procaryotic membranes, while zwitterionic dodecyl phosphocholine (DPC) was used as eukaryotic membrane surrogate. In an aqueous environment, Of-Pis1 adopts a flexible random coil conformation. In DPC and SDS instead, the N-terminal region of Of-Pis1 forms an amphipathic α-helix with the non-polar face in close contact with the micelles. Slower solvent exchange and higher pK_as of the histidine residues in SDS than in DPC suggest that Of-Pis1 interacts more tightly with SDS. Of-Pis1 also binds tightly and structurally perturbs LPS micelles. Of-Pis1 interacts with both Escherichia coli and mammalian cell membranes, but only in the presence of Escherichia coli membranes it populates the helical conformation. Furthermore, ligand-based NMR experiments support a tighter and more specific interaction with bacterial than with eukaryotic membranes. Overall, these data clearly show the selective interaction of this broadly active AMP with bacterial over eukaryotic membranes. The conformational information is discussed in terms of Of-Pis1 amino acid sequence and composition to provide insights useful to design more potent and selective AMPs.     

Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution.

Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. Here we present the high resolution structure of a recombinant LALF. It has a single domain consisting of three alpha-helices packed against a four-stranded beta-sheet. The wedge-shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and we propose that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.     

Protective effects of antimicrobial peptide S-thanatin against endotoxic shock in mice introduced by LPS

Sepsis continues to be a major unresolved medical challenge of the present. Severe sepsis and septic shock are the leading causes of multiple organ failure and mortality in noncoronary intensive care units (ICUs). The primary reason of septic shock is the activation of host effecter cells by endotoxin and lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the key point of treatment is removing LPS. S-thanatin (Ts), an analog of thanatin, was synthesized by substituting the 15th amino acid of threonine with serine, which showed a broad antimicrobial activity against gram-negative and gram-positive bacteria. We have reported its LPS-binding and -neutralizing activity in vitro. The aim of this study is to examine the LPS-neutralizing activities and the protective effects of S-thanatin in vivo. Every mice was injected intraperitoneally with LPS (from Escherichia coli O111:B4) 150 μg before injected intraperitoneally or vena caudalis with 3 mg/kg, 6 mg/kg and 12 mg/kg, and measured endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, as well as lethality. The results showed that S-thanatin can significantly reduce endotoxin and TNF-α level in plasma, at the same time resulting in the highest survival rates.     

A Peptide of Heparin Cofactor II Inhibits Endotoxin-Mediated Shock and Invasive Pseudomonas aeruginosa Infection

Sepsis and septic shock remain important medical problems with high mortality rates. Today''s treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment options are urgently needed to counteract these complex sepsis pathologies. Heparin cofactor II (HCII) has recently been shown to be protective against Gram-negative infections. The antimicrobial effects were mapped to helices A and D of the molecule. Here we show that KYE28, a 28 amino acid long peptide representing helix D of HCII, is antimicrobial against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida albicans. Moreover, KYE28 binds to LPS and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro-inflammatory response, which lead to a significant reduction of mortality. In summary, the peptide KYE28, by simultaneously targeting bacteria and LPS-induced pro-inflammatory responses represents a novel therapeutic candidate for invasive infections.     

Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins

To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.     

Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins

To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.     

Progressive structuring of a branched antimicrobial Peptide on the path to the inner membrane target

In recent years, interest has grown in the antimicrobial properties of certain natural and non-natural peptides. The strategy of inserting a covalent branch point in a peptide can improve its antimicrobial properties while retaining host biocompatibility. However, little is known regarding possible structural transitions as the peptide moves on the access path to the presumed target, the inner membrane. Establishing the nature of the interactions with the complex bacterial outer and inner membranes is important for effective peptide design. Structure-activity relationships of an amphiphilic, branched antimicrobial peptide (B2088) are examined using environment-sensitive fluorescent probes, electron microscopy, molecular dynamics simulations, and high resolution NMR in solution and in condensed states. The peptide is reconstituted in bacterial outer membrane lipopolysaccharide extract as well as in a variety of lipid media mimicking the inner membrane of Gram-negative pathogens. Progressive structure accretion is observed for the peptide in water, LPS, and lipid environments. Despite inducing rapid aggregation of bacteria-derived lipopolysaccharides, the peptide remains highly mobile in the aggregated lattice. At the inner membranes, the peptide undergoes further structural compaction mediated by interactions with negatively charged lipids, probably causing redistribution of membrane lipids, which in turn results in increased membrane permeability and bacterial lysis. These findings suggest that peptides possessing both enhanced mobility in the bacterial outer membrane and spatial structure facilitating its interactions with the membrane-water interface may provide excellent structural motifs to develop new antimicrobials that can overcome antibiotic-resistant Gram-negative pathogens.     

The proinflammatory activity of lipopolysaccharide

Lipopolysaccharide (LPS) is the major component of the outer membranes of the walls of gramnegative bacteria that protects bacterial cells against antibacterial agents. LPS is the heteropolimer consisting of two parts: the hydrophobic lipid A and hydrophilic polisaccharide. After lysis of bacterial cells LPS is released into the circulation and it is able to activate the immunological system by stimulation of monocytes, macrophages, neutrophils, blood platelets and endothelial cells. LPS can bind to plasma proteins and form complexes that increase toxic activity of LPS and affinity of LPS to cell receptors. The activation of immune cells by LPS leads to release of inflammatory mediators: cytokines, chemokines, enzymes, eicosanoids, adhesion agents and free radicals that are responsible for progression of inflammatory reactions and may induce pathophysiological processes including septic shock.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Tandem repeats of Sushi3 peptide with enhanced LPS-binding and -neutralizing activities

Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, was previously shown to bind and neutralize LPS activity. For large-scale production of this peptide and to mimick other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in Escherichia coli. The recombinant tetramer of S3 (rS3-4mer) was purified by anion exchange and digested into monomers (rS3-1mer). Both the rS3-4mer and rS3-1mer were functionally analyzed for their ability to bind LPS by an ELISA-based method and surface plasmon resonance. The LAL inhibition and TNFalpha-release test showed that rS3-1 mer can neutralize the LPS activity as effectively as the synthetic S3 peptide, while rS3-4mer displays an enhanced inhibitory effect on LPS-induced activities. Both recombinant peptides exhibited low cytotoxicity and no haemolytic activity on human cells. This evidence suggests that the recombinant sushi peptides have potential use for the detection, removal of endotoxin and/or anti-endotoxin strategies.     

Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin’s antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.     

Physico-chemical and biophysical study of the interaction of hexa- and heptaacyl lipid A from Erwinia carotovora with magainin 2-derived antimicrobial peptides

The neutralization of endotoxin structures such as the active ‘endotoxic principle’ lipid A by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of Gram-negative bacteria which frequently may lead to the septic shock syndrome. An effective antimicrobial peptide, originally found in the skin of an African frog, is magainin 2. Here, the interaction of magainin 2-amide and a peptide derived thereof, M2V, with chemically defined and homogeneous hexaacyl and heptaacyl lipids A isolated from LPS of Erwinia carotovora, was investigated. By using Fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid A and the conformation of their phosphate groups due to peptide binding was investigated. The former parameter was also determined by using differential scanning calorimetry. The electrophoretic mobility of lipid A aggregates under the influence of the peptides was studied to determine the Zeta potential, and small-angle X-ray scattering was applied for the elucidation of the types of aggregate structures in the absence and presence of the peptides. The lipid A-induced cytokine production in human mononuclear cells shows that the ability of the two peptides to inhibit a tumor necrosis factor-α production correlates with characteristic changes of the biophysical parameters. These are much stronger expressed for the peptide M2V than for magainin 2-amide, which apparently is connected with the higher number of positive as well as more hydrophobic amino acids, leading to a stronger amphiphilicity necessary to neutralize the amphiphilic lipid A aggregates.     

Binding of LL-37 to model biomembranes: Insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X = 0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Galleria mellonella native and analogue peptides Gm1 and ΔGm1. I) Biophysical characterization of the interaction mechanisms with bacterial model membranes

Natural occurring antimicrobial peptides (AMPs) are important components of the innate immune system of animals and plants. They are considered to be promising alternatives to conventional antibiotics. Here we present a comparative study of two synthetic peptides: Gm1, corresponding to the natural overall uncharged peptide from Galleria mellonella (Gm) and ΔGm1, a modified overall positively charged Gm1 variant. We have studied the interaction of the peptides with lipid membranes composed of different kinds of lipopolysaccharides (LPS) and dimyristoylphosphatidylglycerol (DMPG), in some cases also dimyristoylphosphatidylethanolamine (DMPE) as representative lipid components of Gram-negative bacterial membranes, by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Gm1 generates a destabilizing effect on the gel to liquid crystalline phase transition of the acyl chains of the lipids, as deduced from a decrease in the phase transition temperature and enthalpy, suggesting a fluidization, whereas ΔGm1 led to the opposite behavior. Further, FTIR analysis of the functional groups of the lipids participating in the interaction with the peptides indicated a shift in the band position and intensity of the asymmetric PO₂⁻ stretching vibration originating from the lipid phosphate groups, a consequence of the sterical changes in the head group region. Interestingly, FRET spectroscopy showed a similar intercalation of both peptides into the DMPG and LPS, but much less into the DMPE membrane systems. These results are discussed in the light of a possible use of the peptides as antimicrobial and anti-endotoxin drugs.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.