temporin and Hemolysis

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Antimicrobial Peptides; Antineoplastic Agents; Biofilms; Candida albicans; Cell Line, Tumor; Chromobacterium; DNA, Bacterial; Enterococcus faecalis; Erythrocytes; Escherichia coli; Hemolysis; Horses; Humans; Inhibitory Concentration 50; Microbial Sensitivity Tests; Models, Molecular; Pseudomonas aeruginosa; Ranidae; Skin; Staphylococcus aureus

In our previous study, temporin-GHaR (GHaR) showed potent antimicrobial activity with strong hemolytic toxicity. To overcome its weakness, we designed GHaR6R, GHaR7R, GHaR8R, GHaR9R, and GHaR9W by changing the number of positive charges and the hydrophobic surface of GHaR. With the exception of GHaR7R, the hemolytic toxicity of the derived peptides had been reduced, and the antimicrobial activities remained close to the parent peptide (except for GHaR9R). GHaR6R, GHaR7R, GHaR8R, and GHaR9W exhibited a great bactericidal effect on

Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Biofilms; Cell Membrane; Dental Caries; Epithelial Cells; Erythrocytes; Hemolysis; Humans; Microbial Sensitivity Tests; Peptides; Streptococcus mutans

The abuse of antibiotics has led to the emergence of multidrug-resistant bacteria, which is becoming a serious worldwide problem people have to face. In our previous study, temporin-GHa (GHa) cloned from

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biofilms; Computational Biology; Hemolysis; Humans; Microbial Sensitivity Tests; Proteins; Ranidae; Staphylococcus aureus; Structure-Activity Relationship

Previously, we identified a potent antimicrobial analogue of temporin L (TL), [Pro

Topics: Amino Acid Sequence; Anti-Infective Agents; Antimicrobial Cationic Peptides; Candida albicans; Circular Dichroism; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Humans; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Proline; Protein Structure, Secondary; Proteins; Structure-Activity Relationship

In this study we designed and synthesized a new library of antimicrobial peptides correlated to [Pro

Topics: Adult; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Death; Cell Line; Dose-Response Relationship, Drug; Erythrocytes; Fungi; Glycine; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Humans; Microbial Sensitivity Tests; Molecular Structure; Proteins; Structure-Activity Relationship

Antimicrobial peptides from a wide range of amphibian species, especially frogs of the genus Rana, have been characterised and are potential therapeutic agents. Here we describe the isolation, purification, and structural and biological characterisation of three novel antimicrobial peptides from the skin secretions of the black spotted frog, Rana nigromaculata, from Northeastern China. The peptides were identified as belonging to two known families: the temporin, which was first identified in R. nigromaculata from China, and the brevinin-2. Temporin-1RNa and temporin-1RNb both containing three positive charges and have a high potency against microorganisms (MIC: 3.13-8.3 μM against Gram-positive bacteria, 12.5-25.0 μM against Gram-negative bacteria, and 6.25-12.5 μM against Candida albicans) and a high haemolytic activity against human erythrocytes (HC50: 100-150 μM). Brevinin-2RNa contains a single intra-disulphide bridge at the C-terminus that is active towards the tested Gram-positive bacteria but is not active against E. coli and P. aeruginosa. The cDNAs encoding three novel peptide precursors were also subsequently cloned from an R. nigromaculata skin cDNA library and sequenced. The precursors contain 58-72 amino acid residues, which include a conserved signal peptide, acidic propeptide, and the mature temporin-1RNa, temporin-1RNb and brevinin-2RNa. The CD spectra of temporin-1RNa and temporin-1RNb in water, 30 mM SDS and 50 % trifluoroethanol (TFE) indicated that both peptides adopted an aperiodic structure in water and an organised structure with an α-helical conformation in TFE and SDS solution. The conformational transition induced by TFE or SDS reflects the potential ability of temporin-1RNa and temporin-1RNb to interact with anionic membranes.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Bodily Secretions; Candida albicans; China; Circular Dichroism; Cloning, Molecular; DNA, Complementary; Erythrocytes; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Microbial Sensitivity Tests; Molecular Sequence Data; Protein Conformation; Proteins; Ranidae; Sequence Analysis, DNA; Skin

Temporin-1CEb shows antimicrobial activity against Gram-positive bacteria, but its therapeutic potential is limited by its haemolysis. In this study, eight temporin-1CEb analogues with altered cationicities and hydrophobicities were synthesized. Increasing cationicity and amphipathicity by substituting neutral and non-polar amino acid residues on the hydrophilic face of the α-helix by five or six lysines increased antimicrobial potency approximately 10-fold to 40-fold, although when the number of positive charges was increased from +6 to +7, the antimicrobial potency was not additionally enhanced. The substitution of an l-lysine with a d-lysine, meanwhile maintaining the net charge and the mean hydrophobicity values, had only a minor effect on its antimicrobial activity, whereas significantly led a decrease in its haemolytic activity. Of all the peptides, l-K6 has the best potential as an antimicrobial agent because its antimicrobial activity against both Gram-positive and Gram-negative bacteria is substantial, and its haemolytic activity is negligible. l-K6 adopts an α-helix in 50% trifluoroethanol/water and 30 mm SDS solutions. l-K6 killed 99.9% of E. coli and S. aureus at 4× MIC in 60 min, and its postantibiotic effect was >5 h. l-K6 affects the integrity of E. coli and S. aureus plasma membranes by rapidly inducing membrane depolarization.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Bacterial Infections; Hemolysis; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Protein Structure, Secondary; Proteins; Ranidae; Skin

Peptides isolated from natural fonts are the object of several studies aimed at finding new molecules possessing antibacterial activity. We focused our studies on peptides originally isolated from the Royal Jelly, the jelleins and on some analogs having a UV reporter at the N- or C-terminus. We found that jelleins are mainly active against gram-positive bacteria; interestingly, they act in synergy with peptides belonging to the family of temporins such as temporin A and temporin B against Staphylococcus aureus A170 and Listeria monocytogenes.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Fatty Acids; Hemolysis; Listeria monocytogenes; Mice; Microbial Sensitivity Tests; Peptides; Proteins; Staphylococcus aureus

While investigating antimicrobial peptide diversity of Amolops loloensis, five novel antimicrobial peptides belonging to two families were identified from skin secretions of this frog. The first family including two members is esculentin-2-AL (esculentin-2-ALa and -ALb); the second family including three members is temporin-AL (temporin-ALd to -ALf). The family of esculentin-2-AL is composed of 37 amino acid residues (aa); the family of temporin-AL is composed of 16, 13 and 10 aa, respectively. All of these antimicrobial peptides showed antimicrobial activities against tested microorganisms. cDNAs encoding precursors of esculentin-2-ALs and temporin-ALs were cloned from the skin cDNA library of A. loloensis. All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. The antimicrobial peptide family of esculentin-2 is firstly reported in the genus of Amolops. Combined with previous reports, a total of four antimicrobial peptide families have been identified from the genus of Amolops; three of them are also found in the genus of Rana. These results suggest the possible evolutionary connection between the genera Amolops and Rana.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA, Complementary; Female; Gene Library; Glycosides; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Male; Molecular Sequence Data; Peptides; Pregnenolone; Proteins; Rabbits; Ranidae; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Skin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Temporins constitute a family of amphipathic alpha-helical antimicrobial peptides (AMPs) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. We have recently investigated two members of this family, temporin A (TA) and temporin L (TL), because of their different spectra of antimicrobial activity and toxicity. Consequently, we developed new analogues with promising biological activities named Pro(3)-TL and Gln(3)-TA. In this work, we performed a detailed NMR analysis of the new analogues in SDS and DPC micelles, which mimic bacterial and mammalian membranes, respectively. NMR studies reveal that strongly hemolytic Gln(3)-TA was in a stable helical conformation along the entire sequence, while weakly hemolytic but antimicrobial Pro(3)-TL showed conformational averaging at the N-terminus. Furthermore, molecular dynamics (MD) simulations on TL and Pro(3)-TL were performed in explicit water and DPC micelles. Simulations indicated that both peptides prefer a location at the micelle-water interface; however, Phe(1) of strongly hemolytic TL was embedded more in depth into DPC, and only TL caused a significant distortion of the micelle shape. By combining NMR and computational analyses, we obtained a molecular-level resolution of the interactions between TL and its analogues with membrane mimicking micelles.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Glutamine; Gram-Positive Bacteria; Hemolysis; Magnetic Resonance Spectroscopy; Membranes, Artificial; Micelles; Peptides; Proline; Protein Conformation; Protein Structure, Secondary; Proteins; Rana temporaria; Structure-Activity Relationship

Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly(1)-->Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr(6)]-BK. In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis). RT-PCR of brain RNA resulted in the amplification of cDNAs encoding ranatuerin-2TOc and ranatuerin-2TOd that contained the amino acid substitutions Lys(6)-->Arg and Ala(14)-->Thr, respectively compared with ranatuerin-2TOb. cDNAs encoding preprobrevinin-1TOa and preprotemporin-TOa were amplified from brain RNA as well as a second preprotemporin cDNA that contained a 10-nucleotide insertion that introduced a frame shift resulting in a premature stop codon. A cDNA encoding a novel peptide, DK25 (DVNDLKNLCAKTHNLLPMCAMFGKK) was amplified from brain RNA but neither DK25 nor its putative post-translationally modified form, DF22-amide (DVNDLKNLCAKTHNLLPMCAMF.NH(2)) displayed antimicrobial or hemolytic activities.

Topics: Amino Acid Sequence; Amino Acid Substitution; Amphibian Proteins; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Base Sequence; Bradykinin; Brain; Hemolysis; Molecular Sequence Data; Organ Specificity; Protein Isoforms; Protein Precursors; Proteins; Ranidae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin

The emergence of multidrug-resistant (MDR) microorganisms makes it increasingly difficult to treat infections. These infections include those associated with Pseudomonas aeruginosa, which are hard to eradicate, especially in patients with a compromised immune system. Naturally occurring membrane-active cationic antimicrobial peptides (CAMPs) serve as attractive candidates for the development of new therapeutic agents. Amphibian skin is one of the richest sources for such peptides, but only a few studies on their in vivo activities and modes of action have been reported. We investigated (i) the activity and mechanism underlying the killing of short CAMPs from frog skin (e.g., temporins and esculentin fragments) on an MDR clinical isolate of P. aeruginosa and (ii) their in vivo antibacterial activities and modes of action, using the minihost model of Caenorhabditis elegans. Our data revealed that in vivo, both temporin-1Tb and esculentin(1-18) were highly active in promoting the survival of Pseudomonas-infected nematodes, although temporin-1Tb did not show significant activity in vitro under the experimental conditions used. Importantly, esculentin(1-18) permeated the membrane of Pseudomonas cells within the infected nematode. To the best of our knowledge, this is the first report showing the ability of a CAMP to permeate the microbial membrane within a living organism. Besides shedding light on a plausible mode of action of frog skin CAMPs in vivo, our data suggest that temporins and esculentins would be attractive molecules as templates for the development of new therapeutics against life-threatening infections.

Topics: Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Anura; Caenorhabditis elegans; Cells, Cultured; Glycosides; Hemolysis; Humans; Pregnenolone; Proteins; Pseudomonas aeruginosa; Pseudomonas Infections; Skin

Amphibian skin antimicrobial peptides exhibit a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacterium and cytotoxic activity responsible for inhibiting the growth of cancer cells. In this present study, six cDNAs encoding antimicrobial peptide precursors were cloned from the skin of Chinese brown frog, Rana chensinensis by RT-PCR and 3'-RACE procedure and identified as preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, preprobrevinin-1CEa, preprobrevinin-1CEb, and preprochensinin-1, respectively. The nucleotide sequences of cDNA encoding 59-65 amino acid composed of 289-315 bp. Preprotemporin-1CEa, preprotemporin-1CEb and preprotemporin-1CEc are members of temporin family, which usually are short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and preprobrevinin-1CEb were identified as the members of the brevinin-1 family of antimicrobial peptides since both peptides contain "RANA box" that it's responsible for forming Cys-bridged cyclic heptapeptides at the C-terminal region of peptide. The nucleotide acid sequence and the deduced amino acid Sequence of preprochensinin-1 were not found to be identity with any known amphibian skin defensive peptides, so, preprochensinin-1 was identified as a novel peptide precursor. Four of bioactive peptides: temporin-1CEa, temporin-1CEb, brevinin-1CEa and chensinin-1 were synthesized to investigate their antimicrobial, anticancer and haemolysis activities. The results showed that all of the synthesized antimicrobial peptides in this study inhibited the growth of the Gram-positive bacterium, and exhibited the anticancer activity against the growth of MCF-7 cells and HeLa cells. Analysis of the R. chensinensis bioactive peptides and their gene expression will be beneficial for preservation of this species.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Antineoplastic Agents; Base Sequence; Cloning, Molecular; DNA, Complementary; Hemolysis; Molecular Sequence Data; Protein Precursors; Proteins; Ranidae; Skin

In this work, the naturally occurring antimicrobial peptides temporin A (TA) and L (TL) are studied by spectroscopic (CD and NMR) techniques and molecular dynamics simulation. We analyzed the interactions of TA and TL with sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelles, which mimic bacterial and mammalian membranes, respectively. In SDS, the peptides prefer a location at the micelle-water interface; in DPC, they prefer a location perpendicular to the micelle surface, with the N-terminus imbedded in the hydrophobic core. TL shows higher propensity, with respect to TA, in forming alpha-helical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles. Hence, we have proposed a different molecular mechanism underlying the antimicrobial and hemolytic activities of the two peptides. We also designed new analogues of TA and TL and found interesting differences in their efficacy against microbial species and human erythrocytes.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Erythrocytes; Hemolysis; Humans; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Models, Molecular; Phosphorylcholine; Proteins; Sodium Dodecyl Sulfate; Spectrometry, Mass, Fast Atom Bombardment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Peptidomic analysis of an extract of the skins of specimens of Dybowski's brown frog Rana dybowskii Gunther, 1876, collected on Tsushima Island, Japan led to the identification of 10 peptides with differential antibacterial and hemolytic activities. The primary structures of these peptides identified them as belonging to the brevinin-1 (5 peptides) and brevinin-2 (5 peptides) families of antimicrobial peptides. A peptide (FIGPIISALASLFG.NH(2)) with structural similarity to members of the temporin family was also isolated but this component lacked cytolytic activity. Phylogenetic relationships among the Japanese brown frogs (R. dybowskii, R. japonica, R. okinavana, R. ornativentris, R. pirica, R. sakuraii, R. tagoi, and R. tsushimensis) are only incompletely understood. Cladograms based upon maximum parsimony analyses of the brevinin-1 and brevinin-2 amino acid sequences provide strong support for a sister-group relationship between R. dybowskii and R. pirica and somewhat weaker support for a sister-group relationship between R. okinavana and R. tsushimensis. These conclusions are consistent with previous analyses based upon allozyme variations and comparisons of the nucleotide sequences of mitochondrial genes.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Chromatography, High Pressure Liquid; Cytotoxins; Erythrocytes; Escherichia coli; Female; Hemolysis; Humans; Microbial Viability; Molecular Sequence Data; Phylogeny; Protein Isoforms; Proteins; Ranidae; Skin; Staphylococcus aureus; Tissue Extracts

Using RT-PCR, two cDNAs encoding preprotemporins were cloned from a total RNA preparation of the skin of Tago's brown frog Rana tagoi. Preprotemporin-1TGa cDNA directs the synthesis of temporin-1TGa (FLPILGKLLSGIL.NH2) previously isolated from R. tagoi skin. Preprotemporin-1TGb cDNA directs the synthesis of a novel 16-amino-acid-residue peptide (AVDLAKIANKVLSSLF.NH2) that, atypically for members of the temporin family, inhibits the growth of Gram-negative bacteria more effectively than Gram-positive bacteria. Preprotemporin-1TGa mRNA and preprotemporin-1TGb mRNA were not detected in skin prior to the onset of metamorphosis (stage 35) but the levels of the transcripts increased markedly during metamorphosis reaching a maximum at stage 38. Exposure of adult animals to 10(-8) M triiodothyronine (T3) for 72 h enhanced expression of the preprotemporin-1TGb gene (approximately threefold) but did not significantly change the level of expression of the preprotemporin-1TGa gene. Exposure of the animals to 10(-8) M T3 and 10(-6) M bisphenol A, an endocrine disrupting chemical that potently inhibits the action of thyroid hormones (THs), reduced expression of the preprotemporin-1TGb gene by 10-fold and the preprotemporin-1TGa gene by threefold. We propose that T3-stimulated synthesis of antimicrobial peptides is important in protecting the animal against microorganisms, particularly at metamorphosis and during skin moulting, but environmental pollutants can inhibit peptide synthesis and render the animal susceptible to invasion by pathogens.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Base Sequence; Benzhydryl Compounds; Cloning, Molecular; DNA, Complementary; Erythrocytes; Gene Expression Regulation, Developmental; Hemolysis; Humans; Lethal Dose 50; Metamorphosis, Biological; Microbial Sensitivity Tests; Molecular Sequence Data; Phenols; Protein Precursors; Proteins; Ranidae; Triiodothyronine

The temporins are a family of hydrophobic, C-terminally alpha-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS(10)GIL.NH2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG(10)KLLSGIL.NH2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 microM) and producing hemolysis of human erythrocytes (LD50 = 50 microM).

Topics: Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Cloning, Molecular; Codon, Nonsense; DNA, Complementary; Hemolysis; Humans; Molecular Sequence Data; Proteins; Ranidae; Reverse Transcriptase Polymerase Chain Reaction; RNA Precursors; RNA, Messenger; Sequence Analysis; Skin

Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action.

Topics: Adenosine Triphosphate; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Antiprotozoal Agents; Bacteria; Cell Membrane; Cell Membrane Permeability; Erythrocytes; Fluorescent Dyes; Hemolysis; Humans; Leishmania; Membrane Potentials; Organic Chemicals; Peptides; Proteins; Rana temporaria

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Candida albicans; Chromatography, High Pressure Liquid; Drug Resistance, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Microbial Sensitivity Tests; Molecular Sequence Data; Molecular Weight; Peptides; Phylogeny; Proteins; Pseudomonas aeruginosa; Ranidae; Skin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Temporin A, 18 analogs, and a cecropin A-temporin A hybrid peptide were tested with antibiotic sensitive and resistant bacteria, fungi, human erythrocytes, and in clotting assays. Several peptides were active in these assays, and some analogs (D-TA, W1-TA, and Con-L4,G10) may be useful lead compounds for further antibiotics development. The activity of temporin A was found to be dependent upon several of its structural features, including amino acid composition and sequence, chirality, helicity, and positive charge.

Topics: Anti-Bacterial Agents; Antifungal Agents; Antimicrobial Cationic Peptides; Biological Assay; Hemolysis; Humans; Peptides; Proteins; Recombinant Fusion Proteins; Structure-Activity Relationship

Temporin A (TA) and a cecropin A-temporin A hybrid peptide (CATA) were synthesized and assayed for their hemolytic, anticoagulant, and antifungal properties. CATA retains significant antifungal activity, is less hemolytic than TA, and inhibits blood coagulation. These results recommend further studies of the biological activities of CATA.

Topics: Amino Acid Sequence; Amino Acids; Anti-Infective Agents; Antifungal Agents; Antimicrobial Cationic Peptides; Blood Coagulation; Erythrocytes; Hemolysis; Humans; Molecular Sequence Data; Peptide Biosynthesis; Peptides; Protein Structure, Tertiary; Proteins; Prothrombin; Thromboplastin