Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes

Animals as well as plants defend themselves against invading pathogenic microorganisms utilizing cationic antimicrobial peptides, which rapidly kill various microbes without exerting toxicity against the host. Physicochemical peptide-lipid interactions provide attractive mechanisms for innate immunity. Many of these peptides form cationic amphipathic secondary structures, typically alpha-helices and beta-sheets, which can selectively interact with anionic bacterial membranes by the aid of electrostatic interactions. Rapid, peptide-induced membrane permeabilization is an effective mechanism of antimicrobial action. This review article summarizes interactions with lipid bilayers of magainins (alpha-helix) and tachyplesins (beta-sheet) discovered in frog skin and horseshoe crab hemolymph, respectively, as archetypes, emphasizing that the mode of interaction is strongly dependent on the physicochemical properties not only of the peptide, but also of the target membrane.     

Events at the host-microbial interface of the gastrointestinal tract. V. Paneth cell alpha-defensins in intestinal host defense

Host defense of the small intestine is mediated, in part, by antimicrobial peptides, including alpha-defensins. In the small intestine, Paneth cells, specialized secretory epithelial cells located at the base of the crypt invaginations lining the intestinal wall, produce alpha-defensins. The alpha-defensins are cysteine-rich cationic peptides with antibiotic activity against a wide range of bacteria and other microbes. Studies of transgenic and knockout mice have supported a pivotal role of Paneth cell alpha-defensins in protection from bacterial pathogens. New data suggest that deficient expression of Paneth cell alpha-defensins may contribute to the pathophysiology of Crohn's disease, a chronic inflammatory bowel disease.     

An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo

Host defense peptides are key components of the innate immune system, providing multi-facetted responses to invading pathogens. Here, we describe that the peptide GKS26 (GKSRIQRLNILNAKFAFNLYRVLKDQ), corresponding to the A domain of heparin cofactor II (HCII), ameliorates experimental septic shock. The peptide displays antimicrobial effects through direct membrane disruption, also at physiological salt concentration and in the presence of plasma and serum. Biophysical investigations of model lipid membranes showed the antimicrobial action of GKS26 to be mirrored by peptide incorporation into, and disordering of, bacterial lipid membranes. GKS26 furthermore binds extensively to bacterial lipopolysaccharide (LPS), as well as its endotoxic lipid A moiety, and displays potent anti-inflammatory effects, both in vitro and in vivo. Thus, for mice challenged with ip injection of LPS, GKS26 suppresses pro-inflammatory cytokines, reduces vascular leakage and infiltration in lung tissue, and normalizes coagulation. Together, these findings suggest that GKS26 may be of interest for further investigations as therapeutic against severe infections and septic shock.     

Induction of non-lamellar lipid phases by antimicrobial peptides: a potential link to mode of action

Antimicrobial peptides are naturally produced by numerous organisms including insects, plants and mammals. Their non-specific mode of action is thought to involve the transient perturbation of bacterial membranes but the molecular mechanism underlying the rearrangement of the lipid molecules to explain the formation of pores and micelles is still poorly understood. Biological membranes mostly adopt planar lipid bilayers; however, antimicrobial peptides have been shown to induce non-lamellar lipid phases which may be intimately linked to their proposed mechanisms of action. This paper reviews antimicrobial peptides that alter lipid phase behavior in three ways: peptides that induce positive membrane curvature, peptides that induce negative membrane curvature and peptides that induce cubic lipid phases. Such structures can coexist with the bilayer structure, thus giving rise to lipid polymorphism induced upon addition of antimicrobial peptides. The discussion addresses the implications of induced lipid phases for the mode of action of various antimicrobial peptides.     

Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?

The activity of antimicrobial peptides has been shown to depend on the composition of the target cell membrane. The bacterial selectivity of most antimicrobial peptides has been attributed to the presence of abundant acidic phospholipids and the absence of cholesterol in bacterial membranes. The high amount of cholesterol present in eukaryotic cell membranes is thought to prevent peptide-induced membrane disruption by increasing the cohesion and stiffness of the lipid bilayer membrane. While the role of cholesterol on an antimicrobial peptide-induced membrane disrupting activity has been reported for simple, homogeneous lipid bilayer systems, it is not well understood for complex, heterogeneous lipid bilayers exhibiting phase separation (or "lipid rafts"). In this study, we show that cholesterol does not inhibit the disruption of raft-containing 1,2-dioleoyl-sn-glycero-3-phosphocholine:1,2-dipalmitoyol-sn-glycero-3-phosphocholine model membranes by four different cationic antimicrobial peptides, MSI-78, MSI-594, MSI-367 and MSI-843 which permeabilize membranes. Conversely, the presence of cholesterol effectively inhibits the disruption of non-raft containing 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyol-sn-glycero-3-phosphocholine lipid bilayers, even for antimicrobial peptides that do not show a clear preference between the ordered gel and disordered liquid-crystalline phases. Our results show that the peptide selectivity is not only dependent on the lipid phase but also on the presence of phase separation in heterogeneous lipid systems.     

The role of Paneth cells and their antimicrobial peptides in innate host defense

The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.     

Diversity of antimicrobial peptides and their mechanisms of action

Antimicrobial peptides encompass a wide variety of structural motifs. Many peptides have alpha-helical structures. The majority of these peptides are cationic and amphipathic but there are also hydrophobic alpha-helical peptides which possess antimicrobial activity. In addition, some beta-sheet peptides have antimicrobial activity and even antimicrobial alpha-helical peptides which have been modified to possess a beta-structure retain part of their antimicrobial activity. There are also antimicrobial peptides which are rich in a certain specific amino acid such as Trp or His. In addition, antimicrobial peptides exist with thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure which allows them to bind to the membrane interface. Indeed, most antimicrobial peptides interact with membranes and may be cytotoxic as a result of disturbance of the bacterial inner or outer membranes. Alternatively, a necessary but not sufficient property of these peptides may be to be able to pass through the membrane to reach a target inside the cell. The interaction of these peptides with biological membranes is not just a function of the peptide but is also modulated by the lipid components of the membrane. It is not likely that this diverse group of peptides has a single mechanism of action, but interaction of the peptides with membranes is an important requirement for most, if not all, antimicrobial peptides.     

Crystal structures of human alpha-defensins HNP4, HD5, and HD6

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.     

Primate defensins

Defensins are endogenous, cysteine-rich antimicrobial peptides that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: alpha-defensins are most common in neutrophils and Paneth cells of the small intestine; beta-defensins protect the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and theta-defensins, which are expressed only in Old World monkeys, lesser apes and orangutans, are lectins with broad-spectrum antiviral efficacy. Here, their discovery and recent advances in understanding their properties and functions are described.     

Peptide induced demixing in PG/PE lipid mixtures: A mechanism for the specificity of antimicrobial peptides towards bacterial membranes?

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140-9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid-crystalline membranes.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.     

Fc gamma RIIIb allele-sensitive release of alpha-defensins: anti-neutrophil cytoplasmic antibody-induced release of chemotaxins

Antineutrophil cytoplasmic Abs (ANCA) can activate neutrophils in an FcgammaR-dependent manner, but the link between this ANCA-induced effect and mononuclear cell activation with the characteristic granuloma formation of Wegener's granulomatosis is unclear. Human alpha-defensins, small cationic antimicrobial peptides, are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and monocytes. In this study, we quantitated the release of alpha-defensins (human neutrophil peptides 1-3) from human neutrophils after targeted FcgammaR cross-linking (XL). Homotypic XL of FcgammaRIIa, FcgammaRIIIb, or heterotypic XL of both receptors resulted in significant release of alpha-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-proteinase 3). This release of alpha-defensins, as well as of other granule constituents (ANCA targets anti-proteinase 3 and myeloperoxidase and elastase), was significantly greater in donors homozygous for the NA1 allele of FcgammaRIIIb than in donors homozygous for NA2. Interestingly, the ANCA-induced release was completely inhibited by the IgG Fc-binding peptide TG19320, which blocks the IgG-Fc region from binding to FcgammaR. Based on their chemotactic properties, alpha-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma formation. The greater activity of the FcgammaRIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

Hydrophobic interactions modulate antimicrobial peptoid selectivity towards anionic lipid membranes

Hydrophobic interactions govern specificity for natural antimicrobial peptides. No such relationship has been established for synthetic peptoids that mimic antimicrobial peptides. Peptoid macrocycles synthesized with five different aromatic groups are investigated by minimum inhibitory and hemolytic concentration assays, epifluorescence microscopy, atomic force microscopy, and X-ray reflectivity. Peptoid hydrophobicity is determined using high performance liquid chromatography. Disruption of bacterial but not eukaryotic lipid membranes is demonstrated on the solid supported lipid bilayers and Langmuir monolayers. X-ray reflectivity studies demonstrate that intercalation of peptoids with zwitterionic or negatively charged lipid membranes is found to be regulated by hydrophobicity. Critical levels of peptoid selectivity are demonstrated and found to be modulated by their hydrophobic groups. It is suggested that peptoids may follow different optimization schemes as compared to their natural analogues.     

High concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome

alpha-Defensins, antimicrobial peptides localized in neutrophils, participate in tissue damage through their cytotoxic effects in neutrophil-mediated pulmonary diseases. Neutrophils play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS). We measured alpha-defensins levels in plasma and bronchoalveolar lavage fluid (BALF) of ARDS patients to assess the kinetics of alpha-defensins in ARDS. Plasma alpha-defensins levels were higher in ARDS patients than in control subjects, and BALF levels were also higher in ARDS patients than in control subjects. In ARDS, BALF alpha-defensins levels correlated with those of interleukin (IL)-8, and plasma alpha-defensins levels also correlated with Lung Injury Score. Peripheral neutrophil alpha-defensins contents were higher in ARDS than the control. IL-8 dose-dependently stimulated alpha-defensins release from cultured neutrophils and these levels were higher in ARDS than the control. Reverse-phase high performance liquid chromatography showed high plasma levels of pro-defensins, precursors of alpha-defensins from the bone marrow in ARDS, although alpha-defensins in peripheral and BALF neutrophils were mature type. In conclusion, high plasma alpha-defensins in ARDS patients result from the release of pro-defensins from bone marrow, rather than mature alpha-defensins from neutrophils that accumulate in the alveolar space. The alpha-defensins contents of peripheral neutrophils in ARDS are higher and easier to release than control.     

Characterization of luminal paneth cell alpha-defensins in mouse small intestine. Attenuated antimicrobial activities of peptides with truncated amino termini

Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including alpha-defensins, termed cryptdins. Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP(-) mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of alpha-defensins after peptide secretion.     

Membrane selectivity by W-tagging of antimicrobial peptides

A pronounced membrane selectivity is demonstrated for short, hydrophilic, and highly charged antimicrobial peptides, end-tagged with aromatic amino acid stretches. The mechanisms underlying this were investigated by a method combination of fluorescence and CD spectroscopy, ellipsometry, and Langmuir balance measurements, as well as with functional assays on cell toxicity and antimicrobial effects. End-tagging with oligotryptophan promotes peptide-induced lysis of phospholipid liposomes, as well as membrane rupture and killing of bacteria and fungi. This antimicrobial potency is accompanied by limited toxicity for human epithelial cells and low hemolysis. The functional selectivity displayed correlates to a pronounced selectivity of such peptides for anionic lipid membranes, combined with a markedly reduced membrane activity in the presence of cholesterol. As exemplified for GRR10W4N (GRRPRPRPRPWWWW-NH(2)), potent liposome rupture occurs for anionic lipid systems (dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) and Escherichia coli lipid extract) while that of zwitterionic dioleoylphosphatidylcholine (DOPC)/cholesterol is largely absent under the conditions investigated. This pronounced membrane selectivity is due to both a lower peptide binding to the zwitterionic membranes (z≈-8-10mV) than to the anionic ones (z≈-35-40mV), and a lower degree of membrane incorporation in the zwitterionic membranes, particularly in the presence of cholesterol. Replacing cholesterol with ergosterol, thus mimicking fungal membranes, results in an increased sensitivity for peptide-induced lysis, in analogy to the antifungal properties of such peptides. Finally, the generality of the high membrane selectivity for other peptides of this type is demonstrated.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.