New Family Of Antimicrobial Peptides Research Articles

Peptidomic approach identifies cruzioseptins, a new family of potent antimicrobial peptides in the splendid leaf frog, Cruziohyla calcarifer

Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer. These 15 novel antimicrobial peptides of 20–32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD– and the sequence motifs –VALGAVSK– or –GKAAL(N/G/S) (V/A)V– in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77μM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3–fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11μM). CZS-1 reached 100% haemolysis at 120.87μM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use. Biological significanceThrough the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named “Cruzioseptins” and contains cationic amphipathic peptides of 20–32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical application.

C- and N-truncated antimicrobial peptides from LFampin 265 - 284: Biophysical versus microbiology results

Lactoferrin is a glycoprotein with two globular lobes, each having two domains. Since the discovery of its antimicrobial properties, efforts have been made to find peptides derived from this protein showing antimicrobial properties. Most peptides initially studied were derived from Lactoferricin B, obtained from the protein by digestion with pepsin. More recently, a new family of antimicrobial peptides (AMPs) derived from Lactoferrin was discovered by Bolcher et al, and named Lactoferrampin (LFampin). The original sequence of LFampin contained residues 268 – 284 from the N1 domain of Lactoferrin. From this peptide, the Bolscher’s group synthesized a collection of peptides obtained by extension and / or truncation at the C or N-terminal sides, in order to unravel the main structural features responsible for antimicrobial action. Here, we present results for three of these peptides, namely LFampin 265 – 284, LFampin 265 — 280, and LFampin 270 – 284. The peptides were tested against bacteria (E. coli and S. sanguinis), fungi (C. albicans), and model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG), and their mixtures at a ratio of 3 : 1 (DMPC : DMPG (3 : 1)). The ability to adopt a helical conformation was followed by a circular dichroism (CD), and the perturbation of the gel to the liquid-crystalline phase transition of the membrane was characterized by differential scanning calorimetry (DSC). Distinct behavior was observed in the three peptides, both from the microbiology and model membrane studies, with the biophysical results showing excellent correlation with the microbiology activity studies. LFampin 265 – 284 was the most active peptide toward the tested microorganisms, and in the biophysical studies it showed the highest ability to form an α-helix and the strongest interaction with model membranes, followed by LFampin 265 – 280. LFampin 270 – 284 was inactive, showing marginal secondary structure and no interaction with the pathogen model membranes.

Novel family of antimicrobial peptides from the skin of Rana shuchinae

So far numerous antimicrobial peptides have been characterized from amphibians. In this work, a new family of antimicrobial peptides, named shuchin, was purified and characterized from skin secretions of the frog, Rana shuchinae that lives in freezing mountains. Totally two members of shuchin (shuchin 1 and 2) were identified with the amino acid sequence of NALSMPRNKCNRALMCFG and NALSSPRNKCDRASSCFG, respectively. cDNAs encoding shuchins were cloned from the skin cDNA library of R. shuchinae. The precursors of shuchin are composed of 62 amino acid residues including the conserved signal peptides, acidic propieces, and mature antimicrobial peptides. Synthetic shuchins showed strong and broad antimicrobial activities against Gram-positive bacteria ( Staphylococcus aureus, and Bacillus cereus; MICs < 12.5 μg/ml), Gram-negative bacteria ( Escherichia coli, Bacillus dysenteriae, Pseudomonas aeruginosa; most MICs from 3.1 to 12.5 μg/ml), and yeast ( Candida albicans; MICs of 6.25 μg/ml), but no hemolytic activity under the effective concentration, thereby provide more leading templates for designing novel anti-infection agents.

Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris

The present study reports the characterization of Ls-Stylicin1, a novel antimicrobial peptide from the penaeid shrimp, Litopenaeus stylirostris. The predicted mature peptide of 82 residues is negatively charged (theoretical pI=5.0) and characterized by a proline-rich N-terminal region and a C-terminal region containing 13 cysteine residues. The recombinant Ls-Stylicin1 has been isolated in both monomeric and dimeric forms. Both display strong antifungal activity against Fusarium oxysporum (1.25μM<MIC<2.5μM), a pathogenic fungus of shrimp, but lower antimicrobial activity against Gram (−) bacteria, Vibrio sp. (40μM<MIC<80μM). However, rLs-Stylicin1 is able to agglutinate Vibrio penaeicidae in vitro in agreement with its potent LPS-binding activity on immobilized LPS of V. penaeicidae (dissociation constant (Kd) of 9.6×10−8M). This molecule with no evident homology to other hitherto described antimicrobial peptides but identified herein several species of penaeid shrimp is thought to be the first member of a shrimp antimicrobial peptide family, which we termed stylicins.

LFampin Derived Antimicrobial Peptide: Biophysical Characterization and Biological Implications of Composition and Structure

The innate immunity factor lactoferrin harbours two antimicrobial sequences situated in close proximity in the N1-domain, Lactoferricin (LFcin) and Lactoferrampin (LFampin). The more recently discovered LFampin by Jan Bolscher's group contains residues 268-284 from the N1 domain of Lactoferrin. Thereafter, a new family of antimicrobial peptides was obtained from LFampin by extension and/or truncation at the C- or N-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. These related synthetic peptides show broad-spectrum bactericidal activities against a range of Gram-positive and Gram-negative bacteria, as well as fungus. Bioactivity was tested towards pathogenic yeast Candida albicansand model bacteria strains.The biophysical interaction with model membranes was studied by Differential Scanning Calorimetry (DSC), Isothermal Titration Calorimetry (ITC), Fluorescence Spectroscopy, Circular Dichroism, Zeta Potential and SAXD measurements.Results will be presented for one of the peptides of this family, Lfampin 265-284, both regarding bioactivity and interaction with liposomes of DMPC, DMPG and DMPC:DMPG (3:1) as model membranes. Furthermore, the biophysical and biological implications of composition and structure will be discussed.

A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with β-sheet in water, Tris–HCl or Tris–HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor.

PCR-based Gene Synthesis, Molecular Cloning, High Level Expression, Purification, and Characterization of Novel Antimicrobial Peptide, Brevinin-2R, in Escherichia Coli

Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti- H. pylori activity from Rana amphibians.

Toxicity Evaluation to Mice of Phylloseptin-1, an Antimicrobial Peptide from the Skin Secretion of Phyllomedusa hypochondrialis (Amphibia)

The growing resistance of microorganisms to antibiotics has been considered as a global public health problem. Therefore, the search for novel antimicrobial drugs, chemically unrelated to the presently used antibiotics, is urgently needed. Our group has recently characterized a new family of antimicrobial peptides – phylloseptins – isolated from the skin secretion of the South American amphibian Phyllomedusa hypochondrialis, which showed a strong antimicrobial effect against Gram-positive and Gram-negative bacteria. We now investigate the in vivo toxicity of synthetic phylloseptin-1 (PS-1) toward bone marrow, liver, spleen, kidney and lung after endovenous administration to Swiss mice of a bolus dose of 4 mg/kg. Genotoxicity was evaluated by quantifying erythrocyte micronuclei. PS-1-treated mice showed no alteration in the histology of liver, spleen, kidney and lung, as well as of blood biochemistry, as compared to normal controls. Cytotoxicity tests, evaluated either by blood cytometry or bone marrow polychromatophilic erythrocyte index, revealed no deleterious effect of PS-1. Moreover, the peptide showed no toxicity towards bone marrow erythrocytes. We concluded that, in a concentration ten times over that providing antimicrobial effect, synthetic PS-1 showed no in vivo toxicity.

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS–PAGE and Western blotting revealed the production of a ∼6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.

&amp;#952;-Defensins: Cyclic Antimicrobial Peptides Produced by Binary Ligation of Truncated &amp;#945;-Defensins

The first cyclic peptide discovered in animals is an antimicrobial octadecapeptide that is expressed in leukocytes of rhesus monkeys. The peptide, termed rhesus Theta-defensin 1 (RTD-1) is the prototype of a new family of antimicrobial peptides, which like the previously characterized alpha- and beta-defensin families, possesses broad spectrum microbicidal activities against bacteria, fungi, and protects mononuclear cells from infection by HIV-1. The cyclic Theta-defensin structure is essential for a number of its antimicrobial properties, as demonstrated by the markedly reduced microbicidal activities of de-cyclized Theta-defensin analogs. Genetic and biochemical experiments disclosed that the biosynthesis of RTD-1 results from the head-to-tail joining of two nine-amino acid peptides, each of which is donated by a separate precursor polypeptide, which are in fact C-terminally truncated pro-alpha-defensins. Alternate combinations of the two nonapeptides generate two additional macaque Theta-defensins, RTD-2 and RTD-3. Humans do not express Theta-defensin peptides, but mRNAs encoding at least two Theta-defensins are expressed in human bone marrow. However, in each case the open reading frame is interrupted by a stop codon in the signal peptide-coding region. The mature Theta-defensin peptide is a two-stranded beta-sheet that, like the alpha- and beta-defensins, is stabilized by three disulfides. However, the parallel orientation of the Theta-defensin disulfide arrangement allows for substantial flexibility around its short axis. Unlike alpha- and beta-defensins, RTD-1 lacks an amphiphilic topology. This may partially explain the unusual interaction between Theta-defensins and phospholipid bilayers.

Cupiennin 1, a New Family of Highly Basic Antimicrobial Peptides in the Venom of the Spider Cupiennius salei(Ctenidae)

A new family of antimicrobial peptides was isolated from the venom of Cupiennius salei. The peptides were purified to homogeneity, and the sequence of cupiennin 1a was determined by Edman degradation: GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH(2). The amino acid sequences of cupiennin 1b, c, and d were obtained by a combination of sequence analysis and mass spectrometric measurements of comparative tryptic peptide mapping. All peptides consist of 35 amino acid residues and are characterized by a more hydrophobic N-terminal chain region and a C terminus composed preferentially of polar and charged residues. The total charge of all cupiennins calculated under physiological conditions is +8, and their C terminus, formed by a glutamic acid residue, is amidated. Conformational studies of the peptides revealed a high helix forming potential. Antimicrobial assays on bacteria with cupiennin 1a, 1d, and synthesized cupiennins 1a* and 1d* showed minimal inhibitory concentrations for bacteria in the submicromolar range. Their lytic effect on human red blood cells was lower by a factor of 8 to 14 than the highly hemolytic melittin. Cupiennin 1a, 1b, 1d, 1a*, and 1d* showed pronounced insecticidal activity. The immediate biological effects and the structural properties of the isolated cupiennins indicate a membrane-destroying mode of action on prokaryotic as well as eukaryotic cells.

Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge.

Penaeidins are members of a new family of antimicrobial peptides isolated from a crustacean, which present both Gram-positive antibacterial and antifungal activities. We have studied the localization of synthesis and storage of penaeidins in the shrimp Penaeus vannamei. The distribution of penaeidin transcripts and peptides in various tissues reveals that penaeidins are constitutively synthesized and stored in the shrimp haemocytes. It was shown by immunocytochemistry, at both optical and ultrastructural levels, that the peptides are localized in granulocyte cytoplasmic granules. The expression and localization of penaeidins were further analysed in shrimp subjected to microbial challenge. We found that (1) penaeidin mRNA levels decrease in circulating haemocytes in the first 3 hours following stimulation and (2) an increase in plasma penaeidin concentration occurs after microbial challenge, together with (3) a penaeidin immunoreactivity in cuticular tissue, which can be related to the chitin-binding activity we demonstrate here for penaeidins.

A family of antimicrobial peptides is produced by processing of a 7S globulin protein in Macadamia integrifolia kernels.

A new family of antimicrobial peptides has been discovered in Macadamia integrifolia. The first member of this new family to be purified from nut kernels was a peptide of 45 aa residues, termed MiAMP2c. This peptide inhibited various plant pathogenic fungi in vitro. cDNA clones corresponding to MiAMP2c encoded a 666 aa precursor protein homologous to vicilin 7S globulin proteins. The deduced precursor protein sequence contained a putative hydrophobic N-terminal signal sequence (28 aa), an extremely hydrophilic N-proximal region (212 aa), and a C-terminal region of 426 aa which is represented in all vicilins. The hydrophilic portion of the deduced protein contained the sequence for MiAMP2c as well as three additional segments having the same cysteine spacing pattern as MiAMP2c. Each member of the MiAMP2 family (i.e. MiAMP2a, b, c and d) consisted of approximately 50 amino acids and contained a C-X-X-X-C-(10-12)X-C-X-X-X-C motif. Subsequent isolations from seed exudates led to the purification of the predicted family members MiAMP2b and 2d, both of which also exhibited antimicrobial activity in vitro. These results suggest that some vicilins play a role in defence during seed germination.

Penaeidins, a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)

We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimps Penaeus vannamei in which immune response has not been experimentally induced. The three molecules display antimicrobial activity against fungi and bacteria with a predominant activity against Gram-positive bacteria. The complete sequences of these peptides were determined by a combination of enzymatic cleavages, Edman degradation, mass spectrometry, and cDNA cloning using a hemocyte cDNA library. The mature molecules (50 and 62 residues) are characterized by an NH2-terminal domain rich in proline residues and a COOH-terminal domain containing three intramolecular disulfide bridges. One of these molecules is post-translationally modified by a pyroglutamic acid at the first position. Comparison of the data obtained from the cDNA clones and mass spectrometry showed that two of these peptides are probably COOH-terminally amidated by elimination of a glycine residue. These molecules with no evident homology to other hitherto described antimicrobial peptides were named penaeidins.

Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils.

A new family of cysteine-rich antimicrobial peptides from bovine neutrophils was isolated and characterized. Thirteen structurally homologous peptides were purified to homogeneity from a granule-rich cytoplasmic fraction of purified blood neutrophils. The complete sequences of the peptides were determined by a combination of enzymatic digestion, Edman degradation, and additional biochemical characterization of the carboxyl termini. The peptides are characterized by a highly cationic 38-42-residue chain which includes 6 invariantly spaced cysteines which form three disulfides. They share a highly conserved consensus sequence which is also found in a recently described epithelial antimicrobial peptide from bovine trachea. The in vitro antibacterial activities of the 13 neutrophil peptides, determined in assays using Staphylococcus aureus and Escherichia coli as test organisms, demonstrated that each peptide possessed antimicrobial activity, and that several were as active as the most potent neutrophil defensin, rabbit NP-1. Though the structural and functional attributes of the bovine neutrophil peptides are similar to those of defensins, the two peptide families are distinguished by their unique consensus sequences and additionally by differing tridisulfide motifs. We therefore propose that this new defensin-like antimicrobial peptide family be named beta-defensins.