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.     

Effects of topology, length, and charge on the activity of a kininogen-derived peptide on lipid membranes and bacteria

Effects of topology, length, and charge on peptide interactions with lipid bilayers was investigated for variants of the human kininogen-derived peptide HKH20 (HKHGHGHGKHKNKGKKNGKH) by ellipsometry, CD, fluorescence spectroscopy, and z-potential measurements. The peptides display primarily random coil conformation in buffer and at lipid bilayers, and their lipid interaction is dominated by electrostatics, the latter evidenced by higher peptide adsorption and resulting membrane rupture for an anionic than for a zwitterionic membrane, as well as by strongly reduced adsorption and membrane rupture at high ionic strength. At sufficiently high peptide charge density, however, electrostatic interactions contribute to reducing the peptide adsorption and membrane defect formation. Truncating HKH20 into overlapping 10 amino acid peptides resulted in essentially eliminated membrane rupture and in a reduced amount peptide charges pinned at the lipid bilayer. Finally, cyclic HKH20 was found to be less efficient than the linear peptide in causing liposome rupture, partly due to a lower adsorption. Analogous results were found regarding bactericidal effects.     

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.     

Role of membranes in the activities of antimicrobial cationic peptides

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.     

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.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

Absorbance-based assay for membrane disruption by antimicrobial peptides and synthetic copolymers using pyrroloquinoline quinone-loaded liposomes

A simple homogeneous assay for the detection of membrane permeabilization by antimicrobial peptides and synthetic copolymers is described. Liposomes encapsulating pyrroloquinoline quinone (PQQ), the prosthetic group of the apoenzyme glucose dehydrogenase (GDH), are used to detect membrane permeabilization by the antimicrobial peptides MSI-594 and MSI-78 as well as various synthetic antimicrobial copolymers in an optical microwell assay. PQQ-loaded liposomes and the peptide or copolymer are added to wells of a 96-well microtiter plate. If the integrity of the liposome is compromised, the PQQ encapsulated in the liposomes is released and available for activating the apoenzyme. The release of PQQ catalyzes a color change in the presence of apo-GDH, glucose, and the redox dye 1,6-dichlorophenol indophenol (DCPIP) that can be evaluated through a visual color change. For more quantitative measurements, the absorbance change over a 30 min period was measured. The absorbance change is related to the activity and concentration for a given antimicrobial agent. Furthermore, by varying liposome compositions to include cholesterol, the potential toxicity of the peptide or polymer toward mammalian cells can be readily evaluated. The assay is simple and sensitive and will be useful for analyzing the membrane permeation/disruption properties of a host of antimicrobial peptides and synthetic polymers.     

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.     

Membrane-active properties of α-MSH analogs: aggregation and fusion of liposomes triggered by surface-conjugated peptides

Reaction of the melanotropin hormone analogs [Nle⁴,D-Phe⁷]-α-MSH and [Nle⁴,D-Phe⁷]-α-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle⁴,D-Phe⁷]-α-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of α-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a β-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.     

Cell penetrating peptide TAT can kill cancer cells via membrane disruption after attachment of camptothecin

Attachment of traditional anticancer drugs to cell penetrating peptides is an effective strategy to improve their application in cancer treatment. In this study, we designed and synthesized the conjugates TAT-CPT and TAT-2CPT by attaching camptothecin (CPT) to the N-terminus of the cell penetrating peptide TAT. Interestingly, we found that TAT-CPT and especially TAT-2CPT could kill cancer cells via membrane disruption, which is similar to antimicrobial peptides. This might be because that CPT could perform as a hydrophobic residue to increase the extent of membrane insertion of TAT and the stability of the pores. In addition, TAT-CPT and TAT-2CPT could also kill cancer cells by the released CPT after they entered cells. Taken together, attachment of CPT could turn cell penetrating peptide TAT into an antimicrobial peptide with a dual mechanism of anticancer action, which presents a new strategy to develop anticancer peptides based on cell penetrating peptides.     

Cholesterol reduces pardaxin's dynamics—a barrel-stave mechanism of membrane disruption investigated by solid-state NMR

While high-resolution 3D structures reveal the locations of all atoms in a molecule, it is the dynamics that correlates the structure with the function of a biological molecule. The complete characterization of dynamics of a membrane protein is in general complex. In this study, we report the influence of dynamics on the channel-forming function of pardaxin using chemical shifts and dipolar couplings measured from 2D broadband-PISEMA experiments on mechanically aligned phospholipids bilayers. Pardaxin is a 33-residue antimicrobial peptide originally isolated from the Red Sea Moses sole, Pardachirus marmoratus, which functions via either a carpet-type or barrel-stave mechanism depending on the membrane composition. Our results reveal that the presence of cholesterol significantly reduces the backbone motion and the tilt angle of the C-terminal amphipathic helix of pardaxin. In addition, a correlation between the dynamics-induced heterogeneity in the tilt of the C-terminal helix and the membrane disrupting activity of pardaxin by the barrel-stave mechanism is established. This correlation is in excellent agreement with the absence of hemolytic activity for the derivatives of pardaxin. These results explain the role of cholesterol in the selectivity of the broad-spectrum of antimicrobial activities of pardaxin.     

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.     

Evaluation of biosensor surfaces for the detection of microtubule perturbation

Dual polarization interferometry (DPI) and resonant mirror (RM) methods were used to characterize the growth of microtubules (MTs) on biosensor surfaces. The structure and dynamics of MTs play an important role in cell division and are a target for many anti-cancer drugs. Evidence from DPI demonstrated the growth of MTs on streptavidin–biotinylated-tubulin surfaces from the increase in mass and thickness, with a simultaneous decrease in density. The initial increase in thickness of 0.236 nm/min suggested the elongation of protofilaments before they join laterally to form the MT, where the rate of growth increased to 0.436 nm/min. Continuous mass increases were also observed when tubulin was added to a similar underlying RM surface. Tubulin binding to these surfaces was also temperature dependent, increasing the absolute response with MT stabilizers, while inhibiting binding with destabilizers when temperature was changed from 15 to 37 °C. Finally, the initial rates of tubulin assembly (mean ± SD, n = 3) with MT-stabilizer agents were significantly higher at 1.50 ± 0.27 and 1.04 ± 0.13 arcseconds/s, respectively, compared to 0.37 ± 0.11 arcseconds/s for tubulin containing GTP only. In the presence of the MT destabilizers, colchicine and dolastatin 10, the slopes of initial rates were lower than in their absence at 0.05 ± 0.01 and 0.27 ± 0.08 arcseconds/s, respectively. This provides evidence for the ability of surface-based optical sensors to distinguish between MT stabilizers and destabilizers, while also paving the path to develop other methods to screen for MT-perturbing agents using the same underlying surface engineering.     

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the “carpet-model” and thus, provide important insights into their antimicrobial mechanisms.     

LL-37, the only human member of the cathelicidin family of antimicrobial peptides

Antimicrobial peptides and their precursor molecules form a central part of human and mammalian innate immunity. The underlying genes have been thoroughly investigated and compared for a considerable number of species, allowing for phylogenetic characterization. On the phenotypical side, an ever-increasing number of very varied and distinctive influences of antimicrobial peptides on the innate immune system are reported. The basic biophysical understanding of mammalian antimicrobial peptides, however, is still very limited. This is especially unsatisfactory since knowledge of structural properties will greatly help in the understanding of their immunomodulatory functions. The focus of this review article will be on LL-37, the only cathelicidin-derived antimicrobial peptide found in humans. LL-37 is a 37-residue, amphipathic, helical peptide found throughout the body and has been shown to exhibit a broad spectrum of antimicrobial activity. It is expressed in epithelial cells of the testis, skin, the gastrointestinal tract, and the respiratory tract, and in leukocytes such as monocytes, neutrophils, T cells, NK cells, and B cells. It has been found to have additional defensive roles such as regulating the inflammatory response and chemo-attracting cells of the adaptive immune system to wound or infection sites, binding and neutralizing LPS, and promoting re-epthelialization and wound closure. The article aims to report the known biophysical facts, with an emphasis on structural evidence, and to set them into relation with insights gained on phylogenetically related antimicrobial peptides in other species. The multitude of immuno-functional roles is only outlined. We believe that this review will aid the future work on the biophysical, biochemical and immunological investigations of this highly intriguing molecule.     

Dual antifungal properties of cationic antimicrobial peptides polybia-MPI: Membrane integrity disruption and inhibition of biofilm formation

With the increasing emergence of resistant fungi, the discovery and development of novel antifungal therapeutics were urgently needed. Compared with conventional antibiotics, the limited propensity of AMPs to induce resistance in pathogens has attracted great interest. In the present study, the antifungal activity and its mechanism-of-action of polybia-MPI, a cationic peptide from the venom of Social wasp Polybia Paulista was investigated. We demonstrated that polybia-MPI could potently inhibit the growth of Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The 50% inhibitory concentrations (IC₅₀) of Polybia-MPI against cancer cells were much higher than the MICs against the tested C. albicans and C. glabrata cells, indicating that polybia-MPI had high selectivity between the fungal and mammalian cells. Our results also indicated that membrane disturbance mechanism was involved in the antifungal activity. Furthermore, polybia-MPI could inhibit the bio film forming of C. glabrata, which was frequently associated with clinically significant biofilm. These results suggest that polybia-MPI has great advantages in the development of antifungal agents.     

No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism.We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb³⁺-containing liposomes. Upon chelation of Tb³⁺ by an aromatic carboxylic acid, the fluorescence of Tb³⁺ increases many fold. The CPP TAT(44-57)-SAM and TAT(37-53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol.In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44-57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44-57).Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44-57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.     

Action mechanism and structural requirements of the antimicrobial peptides, gaegurins

Gaegurins (GGNs) are a family of cationic, α-helical, antimicrobial peptides that were isolated from a Korean frog, Glandirana emeljanovi (formerly classified as Rana rugosa) and represent one of the structurally well-characterized groups. Among six gaegurins, gaegurin 4 (renamed herein esculentin-2EM), gaegurin 5 (brevinin-1EMa), and gaegurin 6 (brevinin-1EMb) have been investigated comprehensively in terms of structure-activity relationships. In this paper, we first suggest renaming of gaegurins according to a recently raised rule of systematic nomenclature. Then, the current understanding of gaegurins is reviewed by summarizing their structure-activity relationships. In particular competing arguments on gaegurins are synthetically inspected. Finally their action mechanism and structural requirements will be discussed.     

In situ characterization of lipid A interaction with antimicrobial peptides using surface X-ray scattering

Lipid A structure at the air-aqueous interface has been studied using pressure-area isotherm methods coupled with the surface X-ray scattering techniques of X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD). Lipid A monolayers were formed at the air-aqueous interface to represent the lipid moiety of the outer membrane of Gram-negative bacteria. Lipid A structure was characterized at surface pressures between 10 and 35 mN/m. Interactions of α-helical antimicrobial peptides LL-37, SMAP-29 and D2A22 with lipid A monolayers were subsequently studied. Although insertion into the lipid A monolayers was observed with the α-helical peptides, little change was seen from the X-ray data, suggesting that the lipid A hydrocarbon chains are involved in reorientation during insertion and that the hydrocarbon chains have a relatively rigid structure.     

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.