Purification,characterization, and sequencing of a novel type of antimicrobial peptides,Fa-AMP1 and Fa-AMP2, from seeds of buckwheat (Fagopyrum esculentum Moench.)

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 microg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.     

Amino acid sequence and antimicrobial activity of chitin-binding peptides, Pp-AMP 1 and Pp-AMP 2, from Japanese bamboo shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

Purification,Characterization, and Sequencing of a Novel Type of Antimicrobial Peptides,Fa-AMP1 and Fa-AMP2, from Seeds of Buckwheat (Fagopyrum esculentum Moench.)

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 μg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.     

中国对虾PC-Ⅲ系列抗菌肽的分离纯化及活性

以我国主要经济海产品中国对虾（Penus chinensis）为研究对象,通过Sephadex G-50、RP-HPLC等技术分离纯化到PC-Ⅲ系列中国对虾天然抗菌肽。经初步鉴定,该系列抗菌肽对革兰氏阴性和革兰氏阳性菌都表现出程度不一的抑菌活性,且不同程度地影响小白鼠离体回肠肌收缩,但无丝氨酸蛋白酶抑制剂活性。用MALDI-TOF质谱对样品进行分析,检测到分子量分别为1071Da和1311Da的两种抗菌肽。这些抗菌肽对对虾抵御微生物的侵袭具有重要的作用。     

Amino Acid Sequence and Antimicrobial Activity of Chitin-Binding Peptides,Pp-AMP 1 and Pp-AMP 2, from Japanese Bamboo Shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI–TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0–8.9 μg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.     

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.     

Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua)

The epidermal mucus of fish species has been found to contain antimicrobial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albicans. This activity varied significantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed-phase chromatography, and monitoring the antimicrobial activity, we have isolated four cationic proteins from the mucus extract. Using N-terminal and C-terminal amino acid sequence analysis, together with MS, the antimicrobial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod.     

Identification and Characterization of Novel Antibacterial Peptides from Skin Secretions of Euphlyctis cyanophlyctis

In this study, we extracted and purified antimicrobial peptides (AMPs) secreted from skin of Euphlyctis cyanophlyctis using reverse phase-high performance liquid chromatography. Three AMPs were isolated from skin secretions of this frog and sequenced using tandem mass spectrometry. The purified peptides were named buforin-EC (1875.05 ± 0.5 Da), cyanophlyctin (2347.50 ± 0.5 Da) and temporin-ECa (1013.33 ± 0.5 Da). Multiple alignments and homology search showed that buforin-EC, cyanophlyctin and temporin-ECa had a homology of 71.43, 47.1, and 69.23% to buforin II, brevinin-2EC, and temporin-1CSc, respectively. Antimicrobial tests demonstrated that our peptides have a great antimicrobial effect on both gram-positive and gram-negative bacteria. The results indicated that they have an overall minimum inhibitory concentration (MIC) below 13 μM against E. coli. No hemolysis was observed in around of their MIC values. In conclusion, skin secretions of E. cyanophlyctis contain a novel class of AMPs with the proper characteristics.     

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: Polybia-MPI: I D W K K L L D A A K Q I L-NH2 (1654.09 Da), Polybia-CP: I L G T I L G L L K S L-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.     

Antimicrobial compounds from the excretions of surgical maggots, Lucilia sericata (Meigen) (Diptera, Calliphoridae)

Maggots of Lucilia sericata are widely used in the therapy of infected wounds and skin ulcers. Antimicrobial materials released by the insects during their feeding period in order to suppress microbial competitors and potential pathogens play the key role both in the maggots’ survival in their natural habitats (animal corpses) and their therapeutic efficacy. Although the antimicrobial activity of the maggots’ excretion was demonstrated about a hundred years ago, little is known about the nature of its active compounds. We studied the structural characteristics and antimicrobial activities of the compounds released by L. sericata maggots into the environment. To isolate the compounds, excretion was collected from the culture of actively feeding last instar larvae, active compounds were purified using a combination of liquid chromatography and antibacterial growth inhibition assay and characterized by mass spectrometry. Two groups of antibacterial compounds were isolated from the excretion: polypeptides with molecular masses ranging from 6466 to 9025 Da and small molecules with molecular masses ranging from 130 to 700 Da. The polypeptides characterized by the masses of 8882 and 9025 Da and showing selective activity against Gram-negative bacteria correspond well to diptericins, antimicrobial peptides previously found in the hemolymph of Calliphoridae maggots and known to be part of immune response to bacterial pathogens. Other high-molecular compounds with masses 6466, 6633, 5772, and 8631 Da have no clear analogs among antimicrobial peptides present in the hemolymph. The nature of small molecules present in the excretion awaits further study. Thus, the diversity of antimicrobial compounds discovered in Lucilia excretion demonstrates a sophisticated strategy that helps the maggots to fight bacteria and other microorganisms settling their environment. The strategy combines secretion of a set of antibacterial peptides involved in insect immune response as well as molecules which function outside the host organism.     

Purification and Characterization of Two Novel Antimicrobial Peptides Subpeptin JM4-A and Subpeptin JM4-B Produced by Bacillus subtilis JM4

An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subtilis JM4 according to biochemical tests and 16S rDNA sequence analysis. The corresponding antimicrobial peptides were purified to homogeneity by ammonium sulfate precipitation, sequential SP-Sepharose Fast Flow, Sephadex G-25 and C18 reverse-phase chromatography, and in the final purification step, two active fractions were harvested, designated as Subpeptin JM4-A and Subpeptin JM4-B. The molecular weights, determined by mass spectrometry, were 1422.71 Da for Subpeptin JM4-A and 1422.65 Da for Subpeptin JM4-B, respectively. Amino acid sequencing showed that they differed from each other only at the seventh amino acid except for three unidentified residues, and the two peptides had no significant sequence homology to the known peptides in the database, indicating that they are two novel antimicrobial peptides. In addition, characteristic measurements indicated that both peptides had a relatively broad inhibitory spectrum and remained active over a wide pH and temperature range.     

Purification and cDNA Cloning of Antimicrobial Peptides from the Skin Secretion of the Chinese Frog Rana chensinensis

The skin secretions of amphibians are a rich source of bioactive peptides. We isolated chensirin-1 and chensirin-2 from the skin secretion of the Chinese frog Rana chensinensis. Sephadex-G-50 and RP-HPLC were employed to purify these peptides. The amino acid sequences of these peptides were VLPLVGNLLNDLLGE and IIPLPLGYFAKKT, respectively, as determined by Edman degradation. The molecular weights were 1578.7 and 1460.8 Da, respectively, as analyzed by HPLC-ESI-MS. The chensirin cDNA was cloned by 5′ and 3′ amplification of cDNA ends, synthesized and purified. The antibacterial activities of the chensirins were tested using minimum inhibitory concentration, the results indicated that chensirins inhibit the growth of gram-negative and gram-positive bacteria. Among them, chensirin-1 is a novel peptide with a higher antibacterial activity compared to other similar antimicrobial peptides. These low molecular weight peptides with good antimicrobial efficacy are considered potential sources for developing new antimicrobial agents to improve traditional drug resistance.

     

Antimicrobial activity of two peptides casecidin 15 and 17, found naturally in bovine colostrum

Aims:  To isolate and characterize peptides from bovine colostrum with antimicrobial activity.
Methods and Results:  Three peptides were purified from fresh colostrum by a range of chromatographic methods using antimicrobial activity against Escherichia coli DH5α to screen for the most active fractions. Two peptides, with antimicrobial activity, casecidin 17 and casecidin 15, were identical to sequences in the C-terminal of bovine β-casein (YQEPVLGPVRGPFPIIV and YQEPVLGPVRGPFPI) and had corresponding molecular masses of 1881·00 and 1669·06 Da, respectively. The third peptide was the known peptide isracidin which has a mass of 2763·80 Da and sequence of RPKHPIKHQGLPQEVLNENLLRF. Casecidin 17 and casecidin 15 had identical minimal inhibition concentrations (MICs) against E. coli DPC6053 of 0·4 mg ml⁻¹. Structural modelling suggested amphiphilic structures having identical inhibitory and structural properties. The MIC value of isracidin against E. coli DPC6053 was 0·2 mg ml⁻¹.
Conclusions:  This study shows the presence of three antimicrobial peptides in colostrum which may contribute to a bioprotective role to limit pathogen contamination. Furthermore, the discovery of casecidin 17 and 15 may provide the basis for novel antimicrobial peptide design.
Significance and Impact of the Study:  This is the first study to characterize peptides with antimicrobial activity present in fresh bovine colostrum.     

Hadrurin, a new antimicrobial peptide from the venom of the scorpion Hadrurus aztecus.

A new antimicrobial peptide, hadrurin, was isolated from the venom of the Mexican scorpion Hadrurus aztecus, by gel filtration on a Sephadex G-50 column, followed by high performance liquid chromatography. It is a basic peptide composed of 41 amino-acid residues with a molecular mass of 4436 Da, and contains no cysteines. A model of the three-dimensional folding of hadrurin is compatible with that of an amphipatic molecule with two alpha-helical segments. Hadrurin demonstrates antimicrobial activity at low micromolar concentration, inhibiting the growth of bacteria such as: Salmonella thyphi, Klebsiella pneumoniae, Enterococcus cloacae, Pseudomonas aeruginosa, Escherichia coli and Serratia marscences. It also shows cytolytic activity when tested in human erythrocytes. Hadrurin and two analogs (C-terminal amidated, and all D-enantiomer) were chemically synthesized. They were used to study the possible molecular mechanism of action by testing their ability to dissipate the diffusion potential of liposomes of different compositions. The results obtained indicate that there are no specific receptor molecules for the action of hadrurin, and the most probable mechanism is through a membrane destabilization activity. It is surmised that hadrurin is used by the scorpion as both an attack and defense element against its prey and putative invasive microorganisms. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the N-terminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide-lipid interactions.     

Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60

A novel antimicrobial peptide, with molecular mass of 1602.0469Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC(50) values of 7.89, 0.60 and 21.96 μg ml(-1), respectively.     

Molecular cloning, expression, purification, and functional characterization of palustrin-2CE, an antimicrobial peptide of Rana chensinensis

Antimicrobial peptides are effector molecules of the innate immunity of amphibians. Here, one antimicrobial peptide cDNA precursor, prepropalustrin-2CE3, from the tadpole of the Chinese brown frog Rana chensinensis was cloned. The coding sequence corresponding to the mature palustrin-2CE peptide was subcloned into pGEX-6p-1. The soluble GST-palustrin-2CE fusion protein was successfully expressed in the BL21(DE3)pLysS strain at 16 °C, and the proportion of the fusion protein reached 35%-39% of the total cellular protein. After removal of the GST-fusion tag, the purity of the palustrin-2CE obtained by Sephadex G50 chromatography was about 97%. Moreover, the purified palustrin-2CE displayed obviously inhibitory activities against the sensitive bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, and multi-drug resistant S. aureus and E. coli. These findings suggest that the tadpole of the Chinese brown frog is a unique source of antimicrobial peptides and indicates the therapeutic potential of the palustrin-2CE peptide.     

Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent.

Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography (HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P < 0.001) compared with 5.6 mm glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin-releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin-releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.     

Purification and Characterization of Antioxidant Peptides from Leukocyte Extract of Crocodylus siamensis

Antioxidant peptides were isolated from the leukocyte extract of the Siamese crocodile, Crocodylus siamensis. Crocodile leukocyte was extracted by a combination of methods including freeze-thawing, acetic acid extraction and homogenization. The peptides in the leukocyte extract were purified by anion exchange chromatography and reversed phase-high performance liquid chromatography. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was used to evaluate the antioxidant activity of the elution peaks at each purification step. As a result, there were two purified peptides exhibiting strong antioxidant activity in reducing free radicals on DPPH molecules. The amino acid sequences of these peptides were determined by LC-MS/MS as TDVLGLPAK (912.5 Da) and DPNAALPAGPR (1,148.6 Da), and their IC₅₀ values were 153.4 and 95.7 μM, respectively. The results of this study therefore indicate that leukocyte extract of C. siamensis contains peptides with antioxidant activity which could be used as a novel antioxidant.     

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata.

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death.