Calcein Leakage Research Articles

Revisiting Peptide Amphiphilicity for Membrane Pore Formation

It has previously been shown that an amphipathic de novo designed peptide made of 10 leucines and four phenylalanines substituted with crown ethers induces vesicle leakage without selectivity. To gain selectivity against negatively charged dimyristoylphosphatidylglycerol (DMPG) bilayers, one or two leucines of the peptide were substituted with positively charged residues at each position. All peptides induce significant calcein leakage of DMPG vesicles. However, some peptides do not induce significant leakage of zwitterionic dimyristoylphosphatidylcholine vesicles and are thus active against only bacterial model membranes. The intravesicular leakage is induced by pore formation instead of membrane micellization. Nonselective peptides are mostly helical, while selective peptides mainly adopt an intermolecular β-sheet structure. This study therefore demonstrates that the position of the lysine residues significantly influences the secondary structure and bilayer selectivity of an amphipathic 14-mer peptide, with β-sheet peptides being more selective than helical peptides.

Determining the effect of the incorporation of unnatural amino acids into antimicrobial peptides on the interactions with zwitterionic and anionic membrane model systems

Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH2) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, β-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models. These compounds also exhibited very different CD spectra in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG SUVs and LUVs, indicating the formation of different conformations on interaction with the two membrane types. This observation is also supported by ITC and calcein leakage data. ITC data suggested these peptides interact primarily with the surface of zwitterionic LUVs and was further supported by fluorescence experiments where the interactions do not appear to be concentration dependent. In the presence of anionic membranes, the interactions appear more complex and the calorimetric and fluorescence data both imply pore formation is dependent on peptide concentration. Furthermore, evidence suggests that as the length of Spacer# 1 increases the mechanism of pore formation also changes. Based on the observed differences in the mechanisms of interactions with zwitterionic and anionic LUVs these AMPs are potential candidates for further drug development.

Osmotin from Calotropis procera latex: New insights into structure and antifungal properties

This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: α-helix (20%), β-sheet (33%), turned (19%) and unordered (28%), RMSD 1%. CpOsm was stable at up to 75°C, and thermal denaturation (Tm) was calculated to be 77.8°C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins.

PH-dependent mode of antibacterial actions of low molecular weight water-soluble chitosan (LMWSC) against various pathogens

This study examined the antibacterial mode of action of low molecular weight water-soluble chitosan (LMWSC; MW1, MW3, MW5 and MW10) using a combination of approaches, including antibacterial assays, culture in media with different pH values, bactericidal kinetics, cellular leakage measurements, depolarization, and electron microscopy, as well as an in vivo study utilizing a wound infection and wound healing model. The antibacterial activity of LMWSC was related inversely to pH, with higher activities being observed at lower pH values. In addition, evaluation of the effect of bactericidal concentrations of LMWSC on the morphology of Bacillus megaterium and Escherichia coli O–157 revealed that it induces filamentation. Furthermore, the degree of depolarization (in Escherichia coli, pH 7.4) and calcein leakage (lipid composition; L-α-phosphatidylethanolamine (PE)/L-α-phosphatidyl-dl-glycerol (PG)=7/3 (w/w), pH 5.4 and 7.4) were evaluated. Moreover, cells cultured at various pHs were evaluated by confocal microscopy (pH 5.4 and 7.4). These results showed that treatment with LMWSC with different molecular weights had different effects on bacteria. In particular, LMWSC (MW10) cause more visible damage to the bacterial cell membrane than lower molecular weight LMWSC, which is due most likely to the penetration of cells by the lower weight molecules. Interestingly, MW5 was found to attack the membrane and penetrate the cells. In addition, scanning electron microscopy showed that MW10 caused significant morphological changes to the surface of the bacteria. Finally, an in vivo study utilizing a wound infection or healing model showed that LMWSC had the potential for use as a lead compound for the development of a novel anti-infective or healing compound.

Perturbations of model membranes induced by pathogenic dynorphin A mutants causing neurodegeneration in human brain

Several effects of the endogenous opioid peptide dynorphin A (Dyn A) are not mediated through the opioid receptors. These effects are generally excitatory, and result in cell loss and induction of chronic pain and paralysis. The mechanism(s) is not well defined but may involve formation of pores in cellular membranes. In the 17-amino acid peptide Dyn A we have recently identified L5S, R6W, and R9C mutations that cause the dominantly inherited neurodegenerative disorder Spinocerebellar ataxia type 23. To gain further insight into non-opioid neurodegenerative mechanism(s), we studied the perturbation effects on lipid bilayers of wild type Dyn A and its mutants in large unilamellar phospholipid vesicles encapsulating the fluorescent dye calcein. The peptides were found to induce calcein leakage from uncharged and negatively charged vesicles to different degrees, thus reflecting different membrane perturbation effects. The mutant Dyn A R6W was the most potent in producing leakage with negatively charged vesicles whereas Dyn A L5S was virtually inactive. The overall correlation between membrane perturbation and neurotoxic response [3] suggests that pathogenic Dyn A actions may be mediated through transient pore formation in lipid domains of the plasma membrane.

Mechanism of Saporin Entry into the Target Cell Cytosol

Saporin is a type I ribosomal inactivating protein (RIP) derived from the plant Saponaria officinalis. It kills target cells through its RNA N-glycosidase activity, but has low in vivo toxicity because of the absence of a cell-binding subunit. Saporin enters the cell by endocytosis and transverses the endosomal membrane to reach its cytosolic target by a mechanism that remains elusive. We hypothesized that saporin may behave like a metamorphic protein that undergoes conformational switching between membrane-rupturing and enzymatic states, which may be mediated by pH and endosomal membranes. To test this hypothesis, we examined saporin interactions with phospholipid membranes that mimic the charge and fluidity of the endosomes, its membrane rupturing capabilities, and pH-dependent conformational changes in saporin. Our data indicate that a) saporin does bind to membranes composed of 80% DOPC, 5% POPC, 15% POPG, as detected by resonance energy transfer experiments b) binding is stronger at pH 5.5 than 7.0 (KD = 0.4 μM and 1.1 μM, respectively), c) saporin causes calcein leakage from phospholipid vesicles in a dose-dependent manner, and d) saporin undergoes significant conformational changes upon acidification in the presence of membranes. Circular dichroism data show a moderate change in saporin secondary structure upon a pH shift from 7.0 to 5.5 in the absence of membranes, which can be interpreted as an alpha-helix to beta-sheet transition. In contrast, in the presence of membranes the protein undergoes a prominent conformational change by gaining a larger alpha-helical content upon a similar pH shift. Together, these results provide a foundation for understanding the molecular mechanisms of membrane translocation of saporin, and probably other type I RIP proteins, which may acquire distinct membrane rupturing and catalytic activities by means of conformational switching, regulated by environmental factors such as pH and membranes.

Destabilization of artificial biomembrane induced by the penetration of tryptophan

The effect of tryptophan on the membrane stability was studied by using three artificial biological membranes including liposome, Langmuir monolayer and solid supported bilayer lipid membrane (s-BLM) as models. All the results indicate that the penetration of tryptophan can destabilize different artificial biological membranes. The diameter of liposome and the leakage of calcein from liposome increased with the increase of tryptophan concentration because the penetration of tryptophan was beneficial for dehydrating the polar head groups of lipids and the formation of fusion intermediates. π-A isotherms of lecithin on the subphase of tryptophan solution further confirm that tryptophan can penetrate into lipid monolayer and reduce the stability of lipid monolayer. When the concentration of tryptophan increased from 0 to 2 × 10 −3 mol L −1, the limiting molecular area of lecithin increased from 110.5 to 138.5 Å 2, but the collapse pressure of the monolayer decreased from 47.6 to 42.3 mN m −1, indicating the destabilization of lipid monolayer caused by the penetration of tryptophan. The resistance spectra of s-BLM demonstrate that the existence of tryptophan leads to the formation of some defects in s-BLM and the destabilization of s-BLM. The values of electron-transfer resistance and double layer capacitance respectively decreased from 5.765 × 10 6 Ω and 3.573 × 10 −8 F to 1.391 × 10 6 Ω and 3.340 × 10 −8 F when the concentration of tryptophan increased from 0 to 2 × 10 −3 mol L −1. Correspondingly, the breakdown voltage of s-BLM decreased from 2.51 to 1.72 V.

Mechanism of Polymer-Induced Hemolysis: Nanosized Pore Formation and Osmotic Lysis

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.

Single-cell-based measurement of supraphysiological thermal injury in human carcinoma cells utilizing a micropatterned hydrogel chip

Hyperthermia affects certain regulatory proteins, kinases or cyclins, resulting in alternations to the cell cycle and even to apoptosis. Damage to the cell plasma membrane is a key factor in the killing of a cell by hyperthermia. Analysis at the single-cell level is necessary for understanding the fundamental mechanisms of hyperthermia-induced cell death and the generation of thermotolerance in surviving cells. Engineering approaches achieving precise control of cellular micropatterning provide the potential for investigating the mechanisms of thermal injury to cells at the single-cell level. The main purpose of this study is to fabricate a hydrogel chip with microwells for cellular patterning and to demonstrate the feasibility of measurement of supraphysiological thermal injury in human carcinoma cells (HeLa cells) at the single-cell level. To accomplish this, measurement of membrane injury by dye leakage post-thermal insult was performed and reported in this work. A hydrogel chip with microwells with different diameters was fabricated. For cell concentrations at 0.5×106 cells/mL, the occupancy of cells on the microchip with 40 µm microwells was up to 86.6%, a value far higher than that found on the 30 µm microwells (approximately 78.5%). Most microwells of 30 µm in diameter (about 70%) were occupied by a single cell. The fluorescent images showed that calcein leakage occurred when cell membranes were damaged under supraphysiological temperatures between 43 and 50°C. The normalized intensity of calcein decreased to 32% under a supraphysiological temperature of 43°C for 20 min. The intensity of calcein in cells was less than 20% under a supraphysiological temperature of 50°C. The feasibility of the single-cell-based experiment of thermal injury in the microchip with hydrogel microwells was therefore successfully demonstrated.

Endosomal DNA release studies using giant unilamellar vesicles as model endosomal membranes

Endosomal DNA release is one of the main barriers to successful non-viral gene delivery, since the inability of DNA to escape from the endosome at an early stage leads to its degradation through trafficking to the lysosomal compartment. It is therefore essential to understand the interactions between commonly used gene delivery vectors and endosomal membranes. While membrane interactions are often studied using small unilamellar vesicles (SUVs) as model bilayers, it is proposed that giant unilamellar vesicles (GUVs) present more realistic models due to their larger size, their superior lipid packing due to reduced surface curvature and the ability to visualise them using light or confocal microscopy. GUVs composed of a mixture of neutral or neutral and negatively charged lipids, representing early or late stage endosomal membranes respectively were prepared by electroformation in calcein, followed by the addition of cobalt chloride to quench background fluorescence. GUVs were then observed by confocal fluorescence microscopy before and after the addition of lipid:DNA complexes composed of equimolar mixture of dimethyldioctadecylammonium bromide (DDAB) with the helper lipid dioleoylphosphatidyl-ethanolamine (DOPE) incorporating a 10 mol% rhodamine-labelled DOPE at a 4:1 lipid:DNA charge ratio. Furthermore, in order to visualise the DNA in relation to the encapsulated calcein (green) and the lipid (red), 4 ,6-diamidino-2-phenylindole (DAPI) was added to highlight the DNA blue. Both endosomal models formed spherical GUVs approximately 10–90 m in diameter and were visible as green calcein-encapsulating vesicles. Upon the addition of lipid:DNA complexes to the early endosomal model, a large number of GUVs were shown to lose fluorescence due to calcein leakage, which was concentration dependent first order kinetics. This was also associated with visible alignment of the lipid (red) and the DNA (blue) around the GUV with possible pore formation and DNA translocation across the endosomal membrane. When lipid:DNA complexes were added to the late endosomal membrane model (which incorporated a small percentage of anionic lipid), calcein leakage and pore formation on the surface of the GUV membranes were clearly visible. Additionally, and exclusively to this model, however, a high number of GUVs were shown to deform after the addition of the complexes with or without calcein leakage. This was thought to be due to electrostatic interactions between the cationic DDAB and the anionic lipid domains of the endosomal membrane. In conclusion, it is thought that DDAB-DOPE:DNA complexes interact with both early and late endosomal membranes, causing pore formation and DNA translocation across the membrane, however the nature of the interaction changes as the endosomes traffic from early to late stages.

Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

BackgroundPre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen P. aeruginosa. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against P. aeruginosa.ResultsWe show here that the cementoin domain adopts an α-helical conformation both by circular dichroism and nuclear magnetic resonance analyses in the presence of membrane mimetics, a characteristic shared with a large number of linear polycationic antimicrobial peptides. However, pre-elafin/trappin-2 and its domains display only weak lytic properties, as assessed by scanning electron micrography, outer and inner membrane depolarization studies with P. aeruginosa and leakage of liposome-entrapped calcein. Confocal microscopy of fluorescein-labeled pre-elafin/trappin-2 suggests that this protein possesses the ability to translocate across membranes. This correlates with the finding that pre-elafin/trappin-2 and elafin bind to DNA in vitro and attenuate the expression of some P. aeruginosa virulence factors, namely the biofilm formation and the secretion of pyoverdine.ConclusionsThe N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides. However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against P. aeruginosa. Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some P.aeruginosa virulence factors, possibly through acting on intracellular targets.

Structure and mechanism of action of a de novo antimicrobial detergent-like peptide

The K4 peptide (KKKKPLFGLFFGLF) was recently demonstrated to display good antimicrobial activities against various bacterial strains and thus represents a candidate for the treatment of multiple-drug resistant infections. In this study, we use various techniques to study K4 behaviour in different media: water, solutions of detergent micelles, phospholipid monolayers and suspension of phospholipid vesicles. First, self-assembly of the peptide in water is observed, leading to the formation of spherical objects around 10nm in diameter. The addition of micelles induces partial peptide folding to an extent depending on the charge of the detergent headgroups. The NMR structure of the peptide in the presence of SDS displays a helical character of the hydrophobic moiety, whereas only partial folding is observed in DPC micelles. This peptide is able to destabilize the organization of monolayer membranes or bilayer liposomes composed of anionic lipids. When added on small unilamellar vesicles it generates larger objects attributed to mixed lipid–peptide vesicles and aggregated vesicles. The absence of calcein leakage from liposomes, when adding K4, underlines the original mechanism of this linear amphipathic peptide. Our results emphasize the importance of the electrostatic effect for K4 folding and lipid destabilization leading to the microorganisms' death with a high selectivity for the eukaryotic cells at the MIC. Interestingly, the micrographs obtained by electronic microscopy after addition of peptide on bacteria are also consistent with the formation of mixed lipid–peptide objects. Overall, this work supports a detergent-like mechanism for the antimicrobial activity of this peptide.

Influence of the papiliocin peptide derived from Papilio xuthus on the perturbation of fungal cell membranes

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, the antifungal effects and the mechanism of actions of papiliocin were investigated. First of all, papiliocin was shown to exert fungicidal activity. To understand the antifungal mechanism(s), propidium iodide influx into Candida albicans cells, induced by papiliocin, was examined. The result indicated that papiliocin perturbed and disrupted the fungal plasma membrane. Furthermore, calcein leakage from large unilamellar vesicles and rhodamine leakage from giant unilamellar vesicles further confirmed and visualized the membrane-disruptive action of papiliocin in the fungal model membrane, respectively. In summary, the present study suggests that papiliocin exerts its antifungal activity by a membrane-active mechanism and that this peptide can be developed into novel potent antifungal agents.

Candidacidal mechanism of a Leu/Lys‐rich α‐helical amphipathic model antimicrobial peptide and its diastereomer composed of D,L‐amino acids

We investigated the mechanism of candidacidal action of a Lys/Leu-rich α-helical model antimicrobial peptide (K(9)L(8)W) and its diastereomeric peptide (D(9)-K(9)L(8)W) composed of D,L-amino acids. K(9)L(8)W killed completely Candida albicans within 30 min, but D(9)-K(9)L(8)W killed only 72% of C. albicans even after 100 min. Tryptophan fluorescence spectroscopy indicated that the fungal cell selectivity of D(9)-K(9)L(8)W is closely correlated with a selective interaction with the negatively charged PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) phospholipids, which mimic the outer leaflet of the plasma membrane of C. albicans. K(9)L(8)W was able to induce almost 100% calcein leakage from PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) liposomes at a peptide:lipid molar ratio of 1:16, whereas D(9)-K(9)L(8)W caused only 25% dye leakage even at a peptide:lipid molar ratio of 1:2. Confocal laser-scanning microscopy revealed that FITC-labeled D(9)-K(9)L(8)W penetrated the cell wall and cell membrane and accumulated inside the cells, whereas FITC-labeled K(9)L(8)W did not penetrate but associated with the membranes. Collectively, our results demonstrated that the candidacidal activity of K(9)L(8) W and D(9)-K(9)L(8)W may be due to the transmembrane pore/channel formation or perturbation of the fungal cytoplasmic membranes and the inhibition of intracellular functions, respectively. Finally, D(9)-K(9)L(8)W with potent anti-Candida activity but no hemolytic activity may be potentially a useful lead compound for the development of novel antifungal agents.

Interaction of cationic antimicrobial peptides with phospholipid vesicles and their antibacterial activity

We have designed and synthesized a series of cationic α-helical AMPs with improved antibacterial activity and selectivity against a broad spectrum of G(+) and G(−) bacteria. In the current study, we intended to gain further insight into the mechanisms of action between AMPs and cellular membranes using model liposomes of various phospholipid compositions. Circular dichroism measurements showed that AMPs adopted amphipathic α-helical conformation in the presence of negatively charged vesicles (DOPC/DOPG = 1:3), while they were largely unstructured when incubated with neutral vesicles (DOPC). The interaction of AMPs with phospholipid vesicles were further analyzed by calcein leakage experiments. AMPs exhibited weak dye-leakage activity for DOPC (neutral) vesicles, while they effectively induced calcein leakage when interacted with DOPC/DOPG-entrapped vesicles. These results indicated that our newly designed cationic AMPs did show preferences for bacteria-mimicking anionic membranes. All of them exert their cytolytic activity by folding into an amphipathic helix upon selectively binding and insertion into the target membrane, leading to breakdown of the membrane structure, thus causing leakage of cell contents, resulting finally in cell death. Elucidating the mechanism of the membranolytic activity of AMPs may facilitate the development of more effective antimicrobial agents.

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells ( Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH 2) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.

Aggregatibacter Actinomycetemcomitans Leukotoxin Disrupts Membranes By Inducing the Formation of An Inverted Hexagonal Lipid Phase

The leukotoxin (LtxA) secreted by Aggregatibacter actinomycetemcomitans is a member of the repeats-in-toxin (RTX) family, and like the other members of the family, is a virulence protein which destroys host cells. LtxA exhibits specificity to human white blood cells, thereby allowing A. actinomycetemcomitans to flourish in the human upper aerodigestive track. Scanning electron micrographs of LtxA-treated human immune cell lines showed that LtxA induces the formation of large pores, which appear to be caused by membrane bending. To understand the molecular mechanism of this interaction, we analyzed the behavior of LtxA-membrane interactions in model membranes. Freeze-fracture transmission electron microscopy revealed the presence of unique structures, such as nanotubules, in multilamellar liposomes treated with LtxA. Formation of these structures indicated that the toxin acts by bending the membrane, possibly by inducing a bilayer-to-nonbilayer transition. This phase transition was quantified using differential scanning calorimetry, and it was found that LtxA is a potent inverted hexagonal (HII) phase promoter. The relationship between HII phase induction and membrane disruption was determined with a calcein leakage assay. LtxA-induced leakage from calcein-encapsulating liposomes composed of lipids with negative curvature, favoring HII phase formation, was significantly enhanced compared to leakage from liposomes composed of lipids with neutral curvature, favoring bilayer formation. Addition of lipids with positive curvature, inhibiters of HII phase formation, or cholesterol sulfate, a bilayer stabilizer, completely eliminated calcein leakage. It appears that LtxA causes membrane disruption by inducing the formation of the HII phase, and its toxic effect is therefore highly dependent on lipid curvature. Supported by NIH DE09517.

Induction of Liposome Leakage by Photodynamic Action: Dependence on the Kind of Fluorescent Probe

Photosensitized damage to liposome membranes was studied by using different dye-leakage assays based on fluorescence dequenching of a series of dyes upon their release from liposomes. Irradiation of liposomes with red light in the presence of a photosensitizer, trisulfonated aluminum phthalocyanine (AlPcS3), resulted in the pronounced leakage of carboxyfluorescein, but rather weak leakage of sulforhodamin B and almost negligible leakage of calcein from the corresponding dye-loaded liposomes. The photosensitized liposome permeabilization was apparently associated with oxidation of lipid double bonds by singlet oxygen as evidenced by the requirement of unsaturated lipids in the membrane composition for the photosensitized liposome leakage to occur and the sensitivity of the latter to sodium azide. The fluorescence correlation spectroscopy measurements revealed marked permeability of photodynamically induced pores in liposome membranes for such photosensitizers as AlPcS3 and AlPcS4. It was proposed that the difference in permeability of these pores to carboxyfluorescein and calcein was associated with size restriction. Verification of this hypothesis by studying the effect of PEGs of different molecular weights is under way.

Effect of L- to D-Peptide Isomerisation on the Activity of Antimicrobial Peptide Anoplin

Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, Anoplin is the smallest linear α-helical antimicrobial peptide found naturally up to date. It has broad spectrum activity against both Gram-positive and Gram-negative bacteria, and little hemolytic activity toward human erythrocytes (1,2). Previous studies showed that substitution of all amino acids in the peptide with D-isomers can increase the bioavailability and reduce peptide degradation without affecting the antimicrobial properties since the net charge and the hydrophobicity are retained (3). In the present work, two stereoisomers of Anoplin were studied using UV resonance Raman spectroscopy, Langmuir Blodgett, atomic force microscopy, calcein leakage assay and antimicrobial assay. UV resonance Raman data indicate that the two forms of the peptide adopt similar conformations in aqueous buffer and in membrane mimicking solutions. Monolayer isotherms show that D-Anoplin has a lightly greater area per molecule than L-Anoplin. Finally, membrane rupturing ability of both stereoisomers was found to depend strongly on membrane composition.(1) Konno, K., Hisada, M., Fontana, R., Lorenzi, C.C.B., Naoki, H., Itagaki, Y., Miwa, A., Kawai, N., Nakata, Y., Yasuhara, T., Neto, J.R., de Azevedo Jr., W.F., Palma, M.S. and Nakajima, T. (2001) Biochim. Biophys. Acta 1550: 70-80.(2) Ifrah, D., Doisy, X., Ryge, R.S. and Hansen, P.R. (2005) J. Pept. Sci. 11: 113-121.(3) Wade, D., Boman, A., Wahlin, B., Drain, C.M., Andreu, D., Boman, H.G. and Merrifield, R.B. (1990) Proc. Nat. Acad. Sci. 87: 4761-4765.

Interactions of Antimicrobial Peptide Latarcin With Model Cell Membrane

Latarcins are linear antimicrobial peptides purified from the venom of the Lachesana tarabaevi spider. They are highly active against Gram-positive and Gram-negative bacteria with minimum inhibitory concentrations (MIC) at the micromolar level and low hemolytic activity (1, 2). In the present work, a 26 residue peptide Latarcin 2a that adopts a helix-hinge-helix conformation in a membrane mimetic environment (1, 2) was studied as well as a derivative obtained by replacing the Guanine 11 by with Alanine. The interaction of the peptides with phospholipid mono and bilayers were investigated using Langmuir-Blodgett monolayer technique, Atomic Force Microscopy (AFM), calcein leakage assay and UV resonance Raman spectroscopy. Effect of small changes in the primary structure of the peptide on the membrane rupturing activity is discussed.