Bacterial susceptibility and resistance to modelin-5.

Modelin-5 (M5-NH2) killed Pseudomonas aeruginosa with a minimum lethal concentration (MLC) of 5.86 µM and strongly bound its cytoplasmic membrane (CM) with a Kd of 23.5 µM. The peptide adopted high levels of amphiphilic α-helical structure (75.0%) and penetrated the CM hydrophobic core (8.0 mN m-1). This insertion destabilised CM structure via increased lipid packing and decreased fluidity (ΔGmix < 0), which promoted high levels of lysis (84.1%) and P. aeruginosa cell death. M5-NH2 showed a very strong affinity (Kd = 3.5 µM) and very high levels of amphiphilic α-helical structure with cardiolipin membranes (96.0%,) which primarily drove the peptide's membranolytic action against P. aeruginosa. In contrast, M5-NH2 killed Staphylococcus aureus with an MLC of 147.6 µM and weakly bound its CM with a Kd of 117.6 µM, The peptide adopted low levels of amphiphilic α-helical structure (35.0%) and only penetrated the upper regions of the CM (3.3 mN m-1). This insertion stabilised CM structure via decreased lipid packing and increased fluidity (ΔGmix > 0) and promoted only low levels of lysis (24.3%). The insertion and lysis of the S. aureus CM by M5-NH2 showed a strong negative correlation with its lysyl phosphatidylglycerol (Lys-PG) content (R2 > 0.98). In combination, these data suggested that Lys-PG mediated mechanisms inhibited the membranolytic action of M5-NH2 against S. aureus, thereby rendering the organism resistant to the peptide. These results are discussed in relation to structure/function relationships of M5-NH2 and CM lipids that underpin bacterial susceptibility and resistance to the peptide.

PEGylation enhances the antibacterial and therapeutic potential of amphibian host defence peptides.

Aurein 2.1, aurein 2.6 and aurein 3.1 are amphibian host defence peptides that kill bacteria via the use of lytic amphiphilic α-helical structures. The C-terminal PEGylation of these peptides led to decreased antibacterial activity (Minimum Lethal Concentration (MLCs) ↓ circa one and a half to threefold), reduced levels of amphiphilic α-helical structure in solvents (α-helicity ↓ circa 15.0%) and lower surface activity (Δπ ↓ > 1.5 mN m-1). This PEGylation of aureins also led to decreased levels of amphiphilic α-helical structure in the presence of anionic membranes and zwitterionic membranes (α-helicity↓ > 10.0%) as well as reduced levels of penetration (Δπ ↓ > 3.0 mN m-1) and lysis (lysis ↓ > 10.0%) of these membranes. Based on these data, it was proposed that the antibacterial action of PEGylated aureins involved the adoption of α-helical structures that promote the lysis of bacterial membranes, but with lower efficacy than their native counterparts. However, PEGylation also reduced the haemolytic activity of native aureins to negligible levels (haemolysis ↓ from circa 10% to 3% or less) and improved their relative therapeutic indices (RTIs ↑ circa three to sixfold). Based on these data, it is proposed that PEGylated aureins possess the potential for therapeutic development; for example, to combat infections due to multi-drug resistant strains of S. aureus, designated as high priority by the World Health Organization.

Linearized esculentin-2EM shows pH dependent antibacterial activity with an alkaline optimum.

Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 µM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 µM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5-26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3-5.1 mN m-1) and lyse (↑ 15.1-32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1-23) in the N → C direction, with - < µH > increasing overall from circa - 0.8 to - 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

Polarized human cholangiocytes release distinct populations of apical and basolateral small extracellular vesicles.

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2-interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.

Biophysical studies on the antimicrobial activity of linearized esculentin 2EM.

Linearized esculentin 2 EM (E2EM-lin) from the frog, Glandirana emeljanovi was highly active against Gram-positive bacteria (minimum lethal concentration ≤ 5.0 µM) and strongly α-helical in the presence of lipid mimics of their membranes (>55.0%). The N-terminal α-helical structure adopted by E2EM-lin showed the potential to form a membrane interactive, tilted peptide with an hydrophobicity gradient over residues 9 to 23. E2EM-lin inserted strongly into lipid mimics of membranes from Gram-positive bacteria (maximal surface pressure changes ≥5.5 mN m-1), inducing increased rigidity (Cs-1 ↑), thermodynamic instability (ΔGmix < 0 â†’ ΔGmix > 0) and high levels of lysis (>50.0%). These effects appeared to be driven by the high anionic lipid content of membranes from Gram-positive bacteria; namely phosphatidylglycerol (PG) and cardiolipin (CL) species. The high levels of α-helicity (60.0%), interaction (maximal surface pressure change = 6.7 mN m-1) and lysis (66.0%) shown by E2EM-lin with PG species was a major driver in the ability of the peptide to lyse and kill Gram-positive bacteria. E2EM-lin also showed high levels of α-helicity (62.0%) with CL species but only low levels of interaction (maximal surface pressure change = 2.9 mN m-1) and lysis (21.0%) with the lipid. These combined data suggest that E2EM-lin has a specificity for killing Gram-positive bacteria that involves the formation of tilted structure and appears to be primarily driven by PG-mediated membranolysis. These structure/function relationships are used to help explain the pore forming process proposed to describe the membranolytic, antibacterial action of E2EM-lin.

Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 µm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP; increased IL-6, p21, SA-ß-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles (EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to generate normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

Extracellular vesicles in liver pathobiology: Small particles with big impact.

Extracellular vesicles (EVs) are nanometer-sized, membrane-bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell-to-cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano-shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (Hepatology 2016;64:2219-2233).

Low pH Enhances the Action of Maximin H5 against Staphylococcus aureus and Helps Mediate Lysylated Phosphatidylglycerol-Induced Resistance.

Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an improved ability to penetrate (ΔΠ = 6.2 mN m(-1)) and lyse (lysis = 48%) Staphylococcus aureus membrane mimics, which incorporated physiological levels of lysylated phosphatidylglycerol (Lys-PG, 60%), compared to that at pH 7 (ΔΠ = 5.6 mN m(-1) and lysis = 40% at pH 7) where levels of Lys-PG are lower (40%). The peptide therefore appears to have optimal function at pH levels known to be optimal for the organism's growth. MH5 killed S. aureus (minimum inhibitory concentration of 90 µM) via membranolytic mechanisms that involved the stabilization of α-helical structure (approximately 45-50%) and showed similarities to the "Carpet" mechanism based on its ability to increase the rigidity (Cs(-1) = 109.94 mN m(-1)) and thermodynamic stability (ΔGmix = -3.0) of physiologically relevant S. aureus membrane mimics at pH 6. On the basis of theoretical analysis, this mechanism might involve the use of a tilted peptide structure, and efficacy was noted to vary inversely with the Lys-PG content of S. aureus membrane mimics for each pH studied (R(2) ∼ 0.97), which led to the suggestion that under biologically relevant conditions, low pH helps mediate Lys-PG-induced resistance in S. aureus to MH5 antibacterial action. The peptide showed a lack of hemolytic activity (<2% hemolysis) and merits further investigation as a potential template for development as an antistaphylococcal agent in medically and biotechnically relevant areas.

MicroRNAs in the Cholangiopathies: Pathogenesis, Diagnosis, and Treatment.

The cholangiopathies are a group of liver diseases resulting from different etiologies but with the cholangiocyte as the primary target. As a group, the cholangiopathies result in significant morbidity and mortality and represent one of the main indications for liver transplant in both children and adults. Contributing to this situation is the absence of a thorough understanding of their pathogenesis and a lack of adequate diagnostic and prognostic biomarkers. MicroRNAs are small non-coding RNAs that modify gene expression post-transcriptionally. They have been implicated in the pathogenesis of many diseases, including the cholangiopathies. Thus, in this review we provide an overview of the literature on miRNAs in the cholangiopathies and discuss future research directions.

The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

Biophysical investigations with MARCKS-ED: dissecting the molecular mechanism of its curvature sensing behaviors.

Curved membranes are a common and important attribute in cells. Protein and peptide curvature sensors are known to activate signaling pathways, initiate vesicle budding, trigger membrane fusion, and facilitate molecular transport across cell membranes. Nonetheless, there is little understanding how these proteins and peptides achieve preferential binding of different membrane curvatures. The current study is to elucidate specific factors required for curvature sensing. As a model system, we employed a recently identified peptide curvature sensor, MARCKS-ED, derived from the effector domain of the myristoylated alanine-rich C kinase substrate protein, for these biophysical investigations. An atomistic molecular dynamics (MD) simulation suggested an important role played by the insertion of the Phe residues within MARCKS-ED. To test these observations from our computational simulations, we performed electron paramagnetic resonance (EPR) studies to determine the insertion depth of MARCKS-ED into differently curved membrane bilayers. Next, studies with varied lipid compositions revealed their influence on curvature sensing by MARCKS-ED, suggesting contributions from membrane fluidity, rigidity, as well as various lipid structures. Finally, we demonstrated that the curvature sensing by MARCKS-ED is configuration independent. In summary, our studies have shed further light to the understanding of how MARCKS-ED differentiates between membrane curvatures, which may be generally applicable to protein curvature sensing behavior.

The interaction of aurein 2.5 with fungal membranes.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH2) is an antimicrobial peptide, which was seen to have activity against Stachybotris chartarum, Penicillium roseopurpureum and Aspergillus flavus with minimum fungicidal concentrations in the range 250-500 µM. S. chartarum showed enhanced susceptibility to lysis as compared to P. roseopurpureum and A. flavus, (44, 26 and 28 % respectively). Monolayers formed from lipid membrane extracts derived from S. chartarum, P. roseopurpureum and A. flavus showed maximal surface pressure changes of 13.5, 10.3 and 10.2 mN m(-1) respectively. However, aurein 2.5 adopted similar levels of α-helical structure (circa 45 %) in the presence of vesicles formed from membrane lipid extracts derived from all three fungi. These data imply that differential activity is not due to targeting and membrane association but linked to the ability of the bound peptide to lyse the cells. At sterol levels mimetic of eukaryotic systems, high levels of α-helical structure (circa 50 %) were also observed and hence similar binding. However, enhanced sterol levels (>0.6) led to a reduction in monolayer membrane interaction, suggesting that the sterols influence efficacy. Consistent with this suggestion, thermodynamic analysis showed that the peptide was able to destabilise model fungal monolayers, as indicated by negative values of ∆Gmix.

Antimicrobial activity of aurein 2.5 against yeasts.

Fungal infections with multiple resistance to conventional antifungals are increasingly becoming a medical problem, and there is an urgent need for new antifungal compounds with novel mechanisms of action. Here, we show that aurein 2.5, a naturally occurring peptide antibiotic, displays activity against the fungal strains: Rhodotorula rubra and Schizosaccharomyces pombe (MICs < 130 µM). The peptide adopted high levels of membrane-interactive α-helical structure (> 65%) in the presence of lipid membranes derived from these organisms and showed strong propensities to penetrate (π ≥ 13 mN m(-1) ) and lyse them (> 70%). Based on these data, we suggest that aurein 2.5 kills yeasts via membranolytic mechanisms and may act as a template for the development of therapeutically useful antifungal agents.

A novel form of bacterial resistance to the action of eukaryotic host defense peptides, the use of a lipid receptor.

Host defense peptides show great potential for development as new antimicrobial agents with novel mechanisms of action. However, a small number of resistance mechanisms to their action are known, and here, we report a novel bacterial resistance mechanism mediated by a lipid receptor. Maximin H5 from Bombina maxima bound anionic and zwitterionic membranes with low affinity (Kd > 225 µM) while showing a strong ability to lyse (>55%) and penetrate (π > 6.0 mN m(-1)) these membranes. However, the peptide bound Escherichia coli and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) membranes with higher affinity (Kd < 65 µM) and showed a very low ability for bilayer lysis (<8%) and partitioning (π > 1.0 mN m(-1)). Increasing levels of membrane DMPE correlated with enhanced binding by the peptide (R(2) = 0.96) but inversely correlated with its lytic ability (R(2) = 0.98). Taken with molecular dynamic simulations, these results suggest that maximin H5 possesses membranolytic activity, primarily involving bilayer insertion of its strongly hydrophobic N-terminal region. However, this region was predicted to form multiple hydrogen bonds with phosphate and ammonium groups within PE head-groups, which in concert with charge-charge interactions anchor the peptide to the surface of E. coli membranes, inhibiting its membranolytic action.

Multivalency amplifies the selection and affinity of bradykinin-derived peptides for lipid nanovesicles.

The trimer of a bradykinin derivative displayed a more than five-fold increase in binding affinity for phosphatidylserine-enriched nanovesicles as compared to its monomeric precursor. The nanovesicle selection is directly correlated with multivalency, which amplifies the electrostatic attraction. This strategy may lead to the development of novel molecular probes for detecting highly curved membrane bilayers.

MARCKS-ED peptide as a curvature and lipid sensor.

Membrane curvature and lipid composition regulates important biological processes within a cell. Currently, several proteins have been reported to sense and/or induce membrane curvatures, e.g., Synaptotagmin-1 and Amphiphysin. However, the large protein scaffold of these curvature sensors limits their applications in complex biological systems. Our interest focuses on identifying and designing peptides that can sense membrane curvature based on established elements observed in natural curvature-sensing proteins. Membrane curvature remodeling also depends on their lipid composition, suggesting strategies to specifically target membrane shape and lipid components simultaneously. We have successfully identified a 25-mer peptide, MARCKS-ED, based on the effector domain sequence of the intracellular membrane protein myristoylated alanine-rich C-kinase substrate that can recognize PS with preferences for highly curved vesicles in a sequence-specific manner. These studies further contribute to the understanding of how proteins and peptides sense membrane curvature, as well as provide potential probes for membrane shape and lipid composition.

Detection of highly curved membrane surfaces using a cyclic peptide derived from synaptotagmin-I.

The generation of highly curved membranes is essential to cell growth, division, and movement. Recent research in the field is focused to answer questions related to the consequences of changes in the topology of the membrane once it is created, broadly termed as membrane curvature sensing. Most probes that are used to study curvature sensing are intact membrane active proteins such as DP1/Yop1p, ArfGAP1, BAR domains, and Synaptotagmin-I (Syt1). Taking a cue from nature, we created the cyclic peptide C2BL3C based on the membrane penetration C2B loop 3 of Syt1 via "Click" chemistry. Using a combination of spectroscopic techniques, we investigated the peptide-lipid interactions of this peptide with synthetic phospholipid vesicles and exosomes from rat blood plasma. We found that the macrocycle peptide probe was selective for lipid vesicles with highly curved surfaces (d < 100 nm). These results suggested that C2BL3C functions as a selective detector of highly curved phospholipid bilayers.

Constant pressure-controlled extrusion method for the preparation of Nano-sized lipid vesicles.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions, monitor drug delivery, and encapsulation. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle. Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs). In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest. Drug encapsulation can be achieved as reported during liposome formation. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles. Nonetheless, the current liposome preparation technologies (sonication, freeze-and-thaw, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes. Manual extrusion using gas-tight syringes and polycarbonate membranes, is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS), electron microscopy and nanoparticle tracking analysis (NTA) were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

Role of molecular architecture on the relative efficacy of aurein 2.5 and modelin 5.

In order to gain an insight into the mechanism of antimicrobial peptide action, aurein 2.5 and modelin-5 were studied. When tested against Staphylococcus aureus, aurein 2.5 showed approximately 5-fold greater efficacy even though the higher net positive charge and higher helix stability shown by modelin-5 would have predicated modelin-5 to be the more effective antimicrobial. However, in the presence of S. aureus membrane mimics, aurein 2.5 showed greater helical content (75% helical) relative to modelin-5 (51% helical) indicative of increase in membrane association. This was supported by monolayer data showing that aurein 2.5 (6.6mNm(-1)) generated greater pressure changes than modelin-5 (5.3mNm(-1)). Peptide monolayers indicted that modelin-5 formed a helix horizontal to the plane of an asymmetric interface which would be supported by the even distribution of charge and hydrophobicity along the helical long axis and facilitate lysis by non-specific membrane binding. In contrast, a groove structure observed on the surface of aurein 2.5 was predicted to be the cause of enhanced lipid binding (K(d)=75µM) relative to modelin-5 (K(d)=118µM) and the balance of hydrophobicity along the aurein 2.5 long axis supported deep penetration into the membrane in a tilt formation. This oblique orientation generates greater lytic efficacy in high anionic lipid (71%) compared to modelin-5 (32%).

Effect of amidation on the antimicrobial peptide aurein 2.5 from Australian southern bell frogs.

Aurein 2.5 is a naturally C-terminally amidated amphibian antimicrobial peptide. C-terminal amidation can increase efficacy and hence a comparison was made between aurein 2.5-CONH2 and its nonamidated analogue. Amidation of the C-terminal carboxyl of aurein 2.5 enhanced antimicrobial activity 2.5- fold against Klebsiella pneumonia. Our results demonstrate that both peptide analogues had high surface activities (23 mN m-1for aurein 2.5-COOH and 26 mN m-1 aurein 2.5-CONH2). Circular dichroism measurements suggest that the helical content of the amidated form, in the presence of trifluoroethanol, was significantly enhanced (33.66 % for aurein 2.5-COOH and 60.89 % aurein 2.5-CONH2). The interaction of aurein 2.5 with bacterial cell membrane mimics was investigated using Langmuir monolayers. Aurein 2.5-CONH2 induced stable surface pressure changes in monolayers formed from K. pneumonia (circa 4.7 mN m-1), however, lower surface pressure changes were observed for aurein 2.5- COOH (circa 3.8 mN m-1). The data shows that in the case of aurein 2.5, amidation is able to enhance antibacterial activity and it is proposed that the increase in effectiveness is due to stabilization of the α-helical structure at the membrane interface.