Induction, purification and characterization of an antibacterial peptide scolopendrin I from the venom of centipede Scolopendra subspinipes mutilans

The crude venom of the centipede Scolopendra subspinipes mutilans, injected with Escherichia coli K12D31 for 3-4 days showed broad-spectrum antimicrobial activity against Gram-positive. Gram-negative bacteria and fungi. It showed good antibacterial activity against E. coli K12D31 at different temperatures, pH, and ionic strengths. The crude venom was heated at 100 degrees C for 30 min, centrifuged at 10,000 rpm for 30 min at 4 degrees C and the supernatants were obtained, from which an antibacterial fraction having a molecular mass of 3000-5000 Da, was further separated by ultrafiltration. A homogeneous antibacterial peptide named scolopendrin I, having a molecular mass of 4,498 Da, was isolated using cation-exchange chromatography and two steps of reverse-phase high performance liquid chromatography (RP-HPLC). Scolopendrin I did not show any hemolytic and agglutination activities at the concentration below 30 microM.     

Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus

An antibacterial peptide with 16 amino acid residues was found in plasma of the freshwater crayfish, Pacifastacus leniusculus. This peptide, designated astacidin 1, was purified by cation-exchange column chromatography and reverse-phase high performance liquid chromatography. Astacidin 1 has a broad range of antibacterial activity, and it inhibits growth of both Gram-positive and Gram-negative bacteria. The primary sequence of astacidin 1 was FKVQNQHGQVVKIFHH-COOH. The molecular mass was 1945.2 Da, and no carbohydrate-linked amino acid residues could be found by mass spectrometry. A synthetic astacidin 1 resulted in similar activity as the authentic astacidin 1 against Gram-positive bacteria, whereas it had less or no activity against Gram-negative bacteria. Three amino-terminal-truncated synthetic peptides were made; they all showed low activity, suggesting that the amino-terminal part of astacidin 1 contributes to the antibacterial activity. The structure of astacidin 1 based on the CD results showed that it has a beta-sheet structure in citric acid buffer at pH 4, 6, and 8. Cloning of astacidin 1 shows that it is the carboxyl-terminal part of crayfish hemocyanin and that astacidin 1 is produced by a proteolytic cleavage from hemocyanin under acidic conditions. The processing and release of astacidin 1 from hemocyanin is enhanced when crayfish are injected with lipopolysaccharide or glucan.     

Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus.

Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.     

Isolation and Identification of a Novel Inducible Antibacterial Peptide from the Skin Mucus of Japanese Eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

In this study, acetone extracts and acidic extracts were prepared from skin mucus, gill, kidney, liver and spleen of the Japanese eel, Anguilla japonica, and they exhibited different levels of antibacterial activities against three strains of Gram-negative bacteria, Edwardsiella tarda, Aeromonas hydrophila, Aeromonas sp. and one Gram-positive bacterium Micrococcus leteus. The mucus was chosen as the source of antibacterial peptide for further purification of antibacterial peptides. Following the intraperitoneal injection of A. hydrophila, one of the main pathogenic bacteria of Japanese eel and many other fish, a peptide was purified from acetic acid extraction of the skin mucus, by using cationic exchange liquid chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated antibacterial peptide, named as AJN-10, exhibited antibacterial activity against A. hydrophila. The AJN-10 is a heat-tolerant and hydrophilic peptide. The molecular weight of this peptide is 6,044.28 Da, as determined by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The 20 N-terminal amino acid sequences were clarified by Edman degradation, and based on results of homology search by BLAST analysis of the 20 N-terminal sequences, the AJN-10 showed little similarity to other proteins in databases.     

Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.     

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.     

Antimicrobial peptides from the Brazilian frog Phyllomedusa distincta.

Different peptides were purified by chromatographic procedures from the skin-secretory glands of the frog Phyllomedusa distincta. These are the first peptides reported from this frog species. Their primary structure was determined by a combination of automated Edman degradation and mass spectrometry. Peptide Q2 contains 25 amino acid residues, peptide Q1 and L have 28 each, peptide M contains 31, and peptide K has 33 amino acid residues. They all showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria, presenting minimal inhibitory concentrations from 0.6 to 40 microM, when tested against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Peptides K, L, and Q1 were chemically synthesized and shown to be active.     

Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity.

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.     

Purification and antimicrobial activity studies of the N-terminal fragment of ubiquitin from human amniotic fluid

A 4.3-kDa antimicrobial peptide was isolated from human amniotic fluid by dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This peptide, which we named Amniotic Fluid Peptide-1 (AFP-1), possessed antimicrobial activity but lacked hemolytic activity. In addition, AFP-1 potently inhibited the growth of a variety of bacteria (Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus), filamentous fungi (Botrytis cinerea, Aspergillus fumigatus, Neurospora crassa and Fusarium oxysporum) and yeast cells (Candida albicans and Cryptococcus neoformans). Automated Edman degradation showed that the N-terminal sequence of AFP-1 was NH(2)-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-. The partial sequence had 100% homology to the N-terminal sequence of ubiquitin. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the molecular mass of AFP-1 was 4280.2 Da. Our data show an antimicrobial activity of ubiquitin N-terminal derived peptide that makes it suitable for use as an antimicrobial agent.     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Induction, selection and antibacterial activity of the antibacterial peptides from lepldopteran insect cultured cell lines

We induced 3 cell lines that were in vitro cultured from Lepidoptera with heat inactivated Escherichia coil DH5α to stimulate the antibacterial peptide followed by antibacterial activity assay,induction dynamic research and Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) experiment.The antibacterial activity of the induced BTI-Tn-5B1 cell line was the highest,and the antibacterial activity increased gradually to the highest level in 16 hours after stimulation.A new antibacterial peptide with a molecular weight of about 8000 Da was preferentially induced in Trichoplusia ni BTI-Tn-5B1 ceils in 16 hours after stimulation.Antibacterial activity assays indicated that it had inhibition against Staphylococcus aureus,Escherichia coli K12D31 and Salmonella derby.It has especially strong inhibition against Gram-negative bacteria such as Escherichia coli KI2D31 and Salmonella derby.     

Defensin‐like peptide3 from black solder fly: Identification,characterization, and key amino acids for anti‐Gram‐negative bacteria

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.     

九香虫抗菌肽CcAMP1的分离纯化和抗菌活性检测

【目的】从药用昆虫九香虫 Coridius chinensis 中分离纯化抗菌肽,为进一步开发九香虫抗菌肽资源及深入挖掘九香虫的药用功能奠定基础。【方法】用大肠杆菌Escherichia coli 和金黄色葡萄球菌 Staphylococcus aureus 混合物作诱导源刺激九香虫产生抗菌肽,对血淋巴进行提取、凝胶过滤层析、固相萃取及反相色谱纯化,活性组分经质谱测定。对分离得到的这种抗菌肽进行人工合成,并进行抗菌活性检测。【结果】本研究获得一种九香虫抗菌肽CcAMP1,由17个氨基酸残基组成,分子量为1 997.37 u,带1个正电荷,表面有5个疏水氨基酸。对人工合成的CcAMP1进行抗菌活性检测表明,该抗菌肽与九香虫血淋巴一样对金黄色葡萄球菌等革兰氏阳性菌和大肠杆菌等革兰氏阴性菌都有较好的抗菌活性,且对革兰氏阴性菌的抗菌活性更强。【结论】从九香虫中分离得到具有较强抗菌活性的阳离子抗菌肽CcAMP1,有较大的开发利用价值。     

Primary structure and in vitro antibacterial properties of the Drosophila melanogaster attacin C Pro-domain

In Drosophila melanogaster, seven distinct families of antimicrobial peptides with different structures and specificities are synthesized by the fat body and released into the hemolymph during the immune response. Using microscale high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Edman degradation, we have isolated and characterized from immune-challenged Drosophila two novel induced molecules, under the control of the Imd pathway, that correspond to post-translationally modified antimicrobial peptides or peptide fragments. The first molecule is a doubly glycosylated form of drosocin, an O-glycosylated peptide that kills Gram-negative organisms. The second molecule represents a truncated form of the pro-domain of the Drosophila attacin C carrying two post-translational modifications and has significant structural similarities to proline-rich antibacterial peptides including drosocin. We have synthesized this peptide and found that it is active against Gram-negative bacteria. Furthermore, this activity is potentiated when the peptide is used in combination with the Drosophila antimicrobial peptide cecropin A. The synergistic action observed between these two molecules suggests that the truncated post-translationally modified pro-domain of attacin C by itself may play an important role in the antimicrobial defense of Drosophila.     

Enhancing Antibacterial Activity Against Escherichia coli K-12 of Peptide Ib-AMP4 with Synthetic Analogues

A family of Ib-AMP4 peptide analogues was obtained by solid phase synthesis, modifying the net charge and hydrophobicity of C-terminal domain by replacing certain amino acidic residues by arginine and tryptophan. Additionally, disulfide bonds were eliminated by replacing the cysteine residues by methionine, which resulted in a decrease in the number of synthesis byproducts, and consequently diminished the subsequent purification steps. The obtained peptides were purified by RP-HPLC and their molecular mass was determined by MALDI-TOF mass spectrometry. The peptide analogues (IC₅₀ between 1 and 50 μM) presented a higher antibacterial activity against Escherichia coli K-12 than the native peptide (IC₅₀ > 100 μM). The hemolytic activity of the peptide with the highest antibacterial efficacy presented no degradation of erythrocytes for a concentration of 1 μM that corresponds to its IC₅₀ value. The results show that the synthesized peptides are good candidates for the treatment of diseases caused by E. coli.     

A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin

A new potent antibacterial protein, for which we propose the name royalisin, was found in royal jelly of the honeybee Apis mellifera L. and purified to homogeneity for the first time by acid extraction, gel filtration, and reverse-phase high pressure liquid chromatography. The primary structure of royalisin was determined to consist of 51 residues, with three intramolecular disulfide linkages, having a calculated molecular mass of 5523 Da. Royalisin is an amphipathic protein, with the C-terminal half of the molecule being rich in charged amino acids; and it showed extensive sequence homology to two other antibacterial proteins, sapecin from embryonic Sarcophaga peregrina cells and phormicins from Phormia terranovae larvae. Royalisin was found to have potent antibacterial activity against Gram-positive bacteria at low concentrations, but not against Gram-negative bacteria. Royalisin may be involved in a defense system active against bacterial invasion of the honeybee.     

Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure

A cationic peptide, designated tachyplesin, was isolated from acid extracts of horseshoe crab (Tachypleus tridentatus) hemocyte debris. It consists of 17 residues and the structure determined by Edman degradation is: (formula; see text) The carboxyl-terminal end of this peptide was identified as arginine alpha-amide, and the whole sequence including the alpha-amide was also confirmed by fast atom bombardment mass spectrometry, indicating a mass value of 2263. Tachyplesin inhibits growth of both Gram-negative and -positive bacteria at low concentrations and formed a complex with bacterial lipopolysaccharide. Tachyplesin seems likely to act as antimicrobial peptide for self-defense in the horseshoe crab against invading microorganisms.     

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-Trypanosoma cruzi activity without cytotoxicity to mammalian cells

Amphibian skin secretions are known as a rich source of biologically active molecules, most of which are alkaloids, biogenic amines, and peptides. Dermaseptins are a class of antimicrobial peptides present in tree frogs of the Phyllomedusa genus. They are cationic molecules of 28-34 residues that permeabilize the membrane of Gram-positive and Gram-negative bacteria, yeasts, and filamentous fungi, showing little or no hemolytic activity. This work reports the isolation, molecular mass analysis, primary structure determination, biological activities, and potential therapeutic applications of an antimicrobial peptide found in the skin secretion of Phyllomedusa oreades, which is a newly described amphibian species endemic of the Brazilian savanna. DS 01 is a 29-residue-long peptide with a molecular mass of 2793.39 Da showing antibacterial properties against Gram-positive and Gram-negative bacteria in the range of 3-25 microm. Anti-protozoan activity was investigated using T. cruzi in its trypomatigote and epimastigote forms cultivated in both cell culture and blood media. Within 2 h after incubation with DS 01 at a final concentration of approximately 6 microm, no protozoan cells were detected. Two synthetic dermaseptins, described previously by our group and named dermadistinctins K and L (DD K and DD L), also had their anti-Trypanosoma cruzi activity investigated and demonstrated similar properties. Toxicity of DS 01 to mouse erythrocytes and white blood cells was evaluated by means of atomic force microscopy and flow cytometry. No morphological alterations were observed at a lytic concentration of DS 01, suggesting its therapeutic value especially as an anti-T. cruzi agent to prevent infections during blood transfusion.     

Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.