dermaseptin-s and Hemolysis

Dermaseptin-PP is a newly discovered anticancer peptide with a unique antitumour mechanism and remarkable effect. However, this α-helix anticancer peptide risks haemolysis when used at high doses, which limits its further application. This study aims to prepare a pH-responsive liposome, Der-loaded-pHSL, using nanotechnology to avoid the haemolysis risk of Dermaseptin-PP and increase its accumulation in tumour sites to enhance efficacy and reduce toxicity.. The characterisation of Der-loaded-pHSL was carried out employing preparation. The effect of haemolysis and tumour inhibition were investigated by in vitro haemolysis assay and cytotoxicity assay. The cell uptake under different pH conditions was investigated by flow cytometry, and the effect of pH on tumour cell selectivity was evaluated. In order to evaluate the in vivo targeting and antitumour effect of Der-loaded-pHSL, the in vivo distribution experiment and the pharmacodynamic experiment were performed using the nude mouse tumour model.. The preparation method of the Der-loaded-pHSL is simple, and the liposome has good nanoparticle characteristics. When Dermaseptin-PP was prepared as liposome, haemolysis was significantly decreased, and tumour cell inhibition was significantly enhanced. Compared with ordinary liposomes, this change was more significant in Der-loaded-pHSL. The uptake of pH-sensitive liposomes was higher in the simulated acidic tumour microenvironment, and the uptake showed a specific acid dependence. In vivo experiments showed that Der-loaded-pHSL had a significant tumour-targeting effect and could significantly enhance the antitumour effect of Dermaseptin-PP.. Der-loaded-pHSL designed in this study is a liposome with a quick, simple, effective preparation method, which can significantly reduce the haemolytic toxicity of Dermaseptin-PP and enhance its antitumour effect by increasing the tumour accumulation and cell intake. It provides a new idea for applying Dermaseptin-PP and other anticancer peptides with α-helical structure.

Topics: Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Cell Line, Tumor; Hemolysis; Liposomes; Mice; Neoplasms; Tumor Microenvironment

The consistently increasing reports of bacterial resistance and the reemergence of bacterial epidemics have inspired the health and scientific community to discover new molecules with antibacterial potential continuously. Frog-skin secretions constitute bioactive compounds essential for finding new biopharmaceuticals. The exact antibacterial characterization of dermaseptin related peptides derived from Agalychnis annae, is limited. The resemblance in their conserved and functionally linked genomes indicates an unprecedented opportunity to obtain novel bioactive compounds.. In this study, we derived a novel peptide sequence and determined its antibacterial potentials.. Consensus sequence strategy was used to design the novel and active antibacterial peptide named 'AGAAN' from skin secretions of Agalychnis annae. The in-vitro activities of the novel peptide against some bacterial strains were investigated. Time kill studies, DNA retardation, cytotoxicity, betagalactosidase, and molecular computational studies were conducted.. AGAAN inhibited P. aeruginosa, E. faecalis, and S. typhimurium at 20 μM concentration. E. coli and S. aureus were inhibited at 25 μM, and lastly, B. subtilis at 50 μM. Kinetics of inactivation against exponential and stationary growing bacteria was found to be rapid within 1-5 hours of peptide exposure, depending on time and concentration. The peptide displayed weak hemolytic activity between 0.01%-7.31% at the antibacterial concentrations. AGAAN efficiently induced bacterial membrane damage with subsequent cell lysis. The peptide's DNA binding shows that it also targets intracellular DNA by retarding its movement. Our in-silico molecular docking analysis displayed a strong affinity to the bacterial cytoplasmic membrane.. AGAAN exhibits potential antibacterial properties that could be used to combat bacterial resistance.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Anura; Consensus Sequence; DNA; Escherichia coli; Hemolysis; Lipid Bilayers; Microbial Sensitivity Tests; Molecular Docking Simulation; Peptides; Protein Conformation, alpha-Helical; Pseudomonas aeruginosa; Sequence Alignment; Staphylococcus aureus

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antineoplastic Agents; Anura; Biofilms; Candida albicans; Cell Line, Tumor; Cell Membrane Permeability; Cloning, Molecular; Endothelial Cells; Erythrocytes; Gene Expression; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Horses; Humans; Protein Structure, Secondary; Recombinant Proteins; Skin

Dermaseptins are an antimicrobial peptide family widely identified from the skin secretions of phyllomeudusinae frogs. Here, we identify Dermaseptin-PC (DM-PC), from the skin secretion of

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Anura; Bacteria; Biofilms; Candida albicans; Cystic Fibrosis; Erythrocytes; Hemolysis; Horses; Humans; Magnesium Chloride; Skin

Antimicrobial peptides (AMPs) have recently attracted great attention due to their rapid action, broad spectrum of activity, and low propensity of resistance development. The successful application of AMPs in the treatment of intracellular infections, however, remains a challenge because of their low penetration efficiency into the pathogen's intracellular niche. Herein, we report that sub-micrometer-sized crystals of the protein Cry3Aa formed within Bacillus thuringiensis are readily and specifically taken up by macrophages. We demonstrate that these protein crystals efficiently encapsulate a known antileishmanial peptide, dermaseptin S1 (DS1), and thereby promote improved cellular uptake of DS1 and its lysosomal accumulation in macrophages. Notably, this targeted delivery of DS1 results in enhanced in vitro and in vivo antileishmanial activity, as well as reduced toxicity to the host macrophages. These findings suggest that the Cry3Aa crystal can be an effective delivery platform for AMPs to treat intramacrophage infections.

Topics: Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Bacillus thuringiensis Toxins; Bacterial Proteins; Cell Line, Tumor; Drug Delivery Systems; Endotoxins; Female; Hemolysin Proteins; Hemolysis; Humans; Inhibitory Concentration 50; Leishmania; Lysosomes; Macrophages, Peritoneal; Mice, Inbred BALB C

Dermaseptin, an antimicrobial peptide participating in the host defence against pathogens, interacts with the membrane of target cells, leading to membrane permeabilization and eventual cell lysis. Dermaseptin has previously been shown to trigger haemolysis. Prior to haemolysis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase in cytosolic Ca²⁺ activity [(Ca²⁺)](i) and formation of ceramide. This study explored whether dermaseptin modifies [Ca²⁺](i) and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine exposure from annexin-V binding, haemolysis from haemoglobin release, ceramide formation from binding of fluorescent antibodies and [Ca²⁺](i) from Fluo3-fluorescence. A 48-hr exposure to dermaseptin (50 μM) was followed by a significant increase in [Ca²⁺](i), a significant increase ceramide abundance, a significant decrease in forward scatter and a significant increase in annexin-V binding. The annexin-V binding after dermaseptin treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. Dermaseptin triggers eryptosis, an effect at least partially due to entry of extracellular Ca²⁺.

Topics: Amphibian Proteins; Annexin A5; Antimicrobial Cationic Peptides; Apoptosis; Calcium; Cell Membrane; Cell Size; Ceramides; Cytosol; Erythrocytes; Hemolysis; Humans; Phosphatidylserines

The aim of this study was to evaluate the biological activity of nine dermaseptin-S1 (DRS-S1) derivatives (synthesized by solid-phase methods and purified) against different pathogens causing genital infections (Trichomonas vaginalis, Herpes simplex virus, Papillomavirus). The in vitro activity on T. vaginalis was determined by counting the protozoon in a hemocytometer after vital staining with trypan blue; antiviral activity of the compounds was tested on monolayers of Vero cells for Herpes simplex virus-1 (GFP) and on 293TT cells for human papillomavirus (HPV-16) pseudovirions (GFP). The cytotoxicity of the derivatives was assessed by evaluating both the hemolytic activity and the effect on Vero and 293TT cells. The DRS-S1 longer peptides demonstrated a superior activity on T. vaginalis but also a certain cytopathic effect. The compounds with 29 amino acids exhibited activity against the two viruses tested at concentrations not toxic to cells. The results obtained show that some of the synthetic peptides assessed have inhibitory activity against the pathogens tested, indicating a potential for the development of new molecules for use as topical microbicides to prevent the sexual transmission of microorganisms.

Topics: Amphibian Proteins; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Survival; Chlorocebus aethiops; Cottontail rabbit papillomavirus; Erythrocytes; Hemolysis; Humans; Protozoan Infections; Simplexvirus; Trichomonas; Vero Cells

The overlapping biological behaviors between some cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) suggest both common and different membrane interaction mechanisms. We thus explore the capacity of selected CPPs and AMPs to reorganize the planar distribution of binary lipid mixtures by means of differential scanning calorimetry (DSC). Additionally, membrane integrity assays and circular dichroism (CD) experiments were performed. Two CPPs (Penetratin and RL16) and AMPs belonging to the dermaseptin superfamily (Drs B2 and C-terminal truncated analog [1-23]-Drs B2 and two plasticins DRP-PBN2 and DRP-PD36KF) were selected. Herein we probed the impact of headgroup charges and acyl chain composition (length and unsaturation) on the peptide/lipid interaction by using binary lipid mixtures. All peptides were shown to be alpha-helical in all the lipid mixtures investigated, except for the two CPPs and [1-23]-Drs B2 in the presence of zwitterionic lipid mixtures where they were rather unstructured. Depending on the lipid composition and peptide sequence, simple binding to the lipid surface that occur without affecting the lipid distribution is observed in particular in the case of AMPs. Recruitments and segregation of lipids were observed, essentially for CPPs, without a clear relationship between peptide conformation and their effect in the lipid lateral organization. Nonetheless, in most cases after initial electrostatic recognition between the peptide charged amino acids and the lipid headgroups, the lipids with the lowest phase transition temperature were selectively recruited by cationic peptides while those with the highest phase transition were segregated. Membrane activities of CPPs and AMPs could be thus related to their preferential interactions with membrane defects that correspond to areas with marked fluidity. Moreover, due to the distinct membrane composition of prokaryotes and eukaryotes, lateral heterogeneity may be differently affected by cationic peptides leading to either uptake or/and antimicrobial activities.

Topics: Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Calorimetry, Differential Scanning; Carrier Proteins; Cell Membrane Permeability; Cell Survival; Cell-Penetrating Peptides; CHO Cells; Circular Dichroism; Cricetinae; Cricetulus; Eye Proteins; Hemolysis; Membrane Lipids; Membranes; Microbial Sensitivity Tests; Nerve Tissue Proteins; Peptides; Protein Conformation; Rats; Structure-Activity Relationship

Truncation and acylation were combined to investigate the broad-spectrum bactericidal and hemolytic peptide S4(1-15). Substitution of up to seven residues with dodecanoic acid (C(12)) gradually led to specific antipseudomonal activity: out of 40 bacterial strains tested in vitro, C(12)-S4(8-15) displayed similar minimal inhibitory concentrations (MICs) as S4(1-15) against Pseudomonas aeruginosa sp. (identical MIC(90)) but was practically inactive against most other bacteria or erythrocytes. Surface plasmon resonance and isothermal titration calorimetry experiments revealed the binding properties of S4(1-15) to be consistent with its nonselective activities, while discriminative activities of C(12)-S4(8-15) correlated with high binding affinity to a membrane containing pseudomonal lipopolysaccharides and with lower affinities to membranes containing nonpseudomonal lipopolysaccharides or cholesterol. Various mechanistic studies failed to detect significant differences in secondary structure, bactericidal kinetics, or ability to perturb the cytoplasmic membrane, pointing to a similar mode of action.

Topics: Acylation; Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Membrane; Hemolysis; Humans; Lipopolysaccharides; Microbial Sensitivity Tests; Pseudomonas; Structure-Activity Relationship

Analysis of the Schistosoma mansoni peptidome for immunomodulatory molecules by solvent extraction and reverse-phase HPLC revealed a 27-amino-acid residue peptide from an extract of cercariae. Using matrix-assisted, laser desorption-ionization, time-of-flight mass spectrometry, the peptide yielded a protonated molecular ion [M + H]+ of m/z 2789. The unequivocal sequence was deduced by automated Edman degradation as: DLWNSIKDMAAAAGRAALNAVTGMVNQ. The peptide exhibited an 80.76% identity with dermaseptin 3.1 from the leaf frog Agalychnis annae, and was therefore named Schistosoma mansoni dermaseptin-like peptide (SmDLP). Immunocytochemical staining using a primary antidermaseptin B2 antibody located SmDLP in acetabular glands of cercariae, in and around schistosomula, and in adult worms and their eggs. Dot-blotting confirmed its presence in extracts (cercariae and worms) and excretion/secretion (E/S) products (transforming cercariae and eggs). This was corroborated by use of a MALDI-ToF spectra database of E/S products from cercariae. Functional characterization of the peptide indicated that SmDLP had typical amphipathic antimicrobial peptide properties, i.e., the ability to lyse human erythrocytes causing a decrease in the levels of nitric oxide produced by monocytic cells. This last function strongly suggests that SmDLP plays a vital role in the parasite's immunoevasion strategy. The possibility that schistosomes acquired this gene from amphibians has been discussed by constructing a phylogenetic tree.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Biomphalaria; Chromatography, High Pressure Liquid; Consensus Sequence; Conserved Sequence; Helminth Proteins; Hemolysis; Hydrophobic and Hydrophilic Interactions; Immunoblotting; Immunohistochemistry; Male; Mice; Molecular Sequence Data; Protein Structure, Secondary; Schistosoma mansoni; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship

To understand relationships between membrane-binding properties of cytolytic peptides and resulting cytotoxicity, we investigated interactions of dermaseptin analogues with model bilayers by means of surface plasmon resonance. First, we tested the system by comparing two native dermaseptins, S1 and S4, whose binding properties were previously characterized in different experimental systems. Validation experiments revealed deviations from the one-to-one interaction model and indicated the binding to proceed by a two-stage mechanism. By calculation of apparent affinity constants and individual affinities for both steps of the interaction, the biosensor technology was able to distinguish between surface-bound peptides that subsequently penetrated into the bilayer and peptides that remained essentially superficially bound. This data interpretation was sustained after analysis of a series of dermaseptin S4 derivatives whose binding data were compared with cytotoxicity, revealing cytolytic activity to correlate mainly with insertion affinity. The data indicate that the potency of highly cytolytic peptides such as K(4)K(20)-S4 is not due to the highest membrane adhesion affinity but to the highest propensity for the inserted state. Similarly, truncated derivatives of 16, 13, and 10 residues showed a progressive reduction in cytotoxicity that best correlated with progressive reduction in insertion affinity. Support for the adhesion versus inserted states was provided by proteolytic experiments with RBC-bound peptides that demonstrated K(4)K(20)-S4 to be protected from enzymatic cleavage, unlike its 13-mer derivative. Overall, using the two-stage model proved instrumental in investigating membrane-binding properties of antimicrobial peptides and capable of explaining the cytolytic properties of closely related analogues.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Carrier Proteins; Cytotoxins; Erythrocyte Membrane; Hemolysis; Humans; Kinetics; Leishmania major; Lipid Bilayers; Models, Chemical; Molecular Sequence Data; Phosphatidic Acids; Phosphatidylcholines; Protein Binding; Surface Plasmon Resonance

The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. Both DS4 and DS3 inhibited the parasite's ability to incorporate [3H]hypoxanthine. However, while DS4 was toxic toward both the parasite and the host erythrocyte, DS3 was toxic only toward the intraerythrocytic parasite. To gain insight into the mechanism of this selective cytotoxicity, we labeled the peptides with fluorescent probes and investigated their organization in solution and in membranes. In Plasmodium-infected cells, rhodamine-labeled peptides interacted directly with the intracellular parasite, in contrast to noninfected cells, where the peptides remained bound to the erythrocyte plasma membrane. Binding experiments to phospholipid membranes revealed that DS3 and DS4 had similar binding characteristics. Membrane permeation studies indicated that the peptides were equally potent in permeating phosphatidylserine/phosphatidylcholine vesicles, whereas DS4 was more permeative with phosphatidylcholine vesicles. In aqueous solutions, DS4 was found to be in a higher aggregation state. Nevertheless, both DS3 and DS4 spontaneously dissociated to monomers upon interaction with vesicles, albeit with different kinetics. In light of these results, we propose a mechanism by which dermaseptins permeate cells and affect intraerythrocytic parasites.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Chromatography, High Pressure Liquid; Erythrocytes; Hemolysis; Humans; Microscopy, Video; Molecular Sequence Data; Peptides; Plasmodium falciparum; Spectrometry, Fluorescence

Dermaseptin, a 34-amino acid residue cationic peptide, was recently shown to inhibit the growth of pathogenic fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. To improve our understanding of the mechanism by which dermaseptin exerts its potent antimicrobial action, a series of either NH2- or COOH-terminally truncated analogs was synthesized. These analogs were evaluated for their ability to inhibit the growth of various pathogenic agents in culture medium. Dermaseptin exerted a lytic action upon bacteria, protozoa, yeasts, and filamentous fungi at micromolar concentrations. No inhibition of proliferation was observed with human KB cells, and dermaseptin did not lyse guinea pig lymphocytes or rabbit erythrocytes at doses up to 200 micrograms/ml. Shortening the peptide chain of dermaseptin to dermaseptin-(3-34) slightly reduced the activity of the peptide, while further reduction of the chain length to residues 14-34, 16-34, 20-34, and 28-34 yielded peptide derivatives devoid of antimicrobial activity. On the other hand, lengthening the peptide chain starting from residues 1-4 to residues 1-8 and 1-18 led to a progressive recovery of the activity of the parent molecule. Whereas the central core of dermaseptin (residues 10-19) was virtually inactive, alteration of the COOH-terminal carboxylic group of dermaseptin-(1-18) to a carboxamide yielded a peptide exhibiting enhanced antimicrobial potency, yet displaying even less in vitro toxicity compared with dermaseptin. Overall, the data indicate that molecular elements responsible for the exceptional antimicrobial potency of dermaseptin are to be traced to the NH2-terminal alpha-helical amphipathic segment spanning residues 1-18 of the molecule. Dermaseptin-(1-18)-NH2 may therefore be considered as a useful and highly tractable tool for identifying key features responsible for membrane permeabilization and as a starting point for the design of new therapeutic agents.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Cell Survival; Circular Dichroism; Drug Stability; Erythrocytes; Fungi; Guinea Pigs; Hemolysis; Humans; KB Cells; Lymphocytes; Microbial Sensitivity Tests; Models, Biological; Molecular Sequence Data; Peptides; Protein Binding; Protein Conformation; Protein Structure, Secondary; Rabbits