Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

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.     

Direct antimicrobial activities of PR-bombesin

PR-bombesin is a bombesin-like peptide derived from the skin of the Chinese red belly toad, Bombina maxima. The 8-residue segment of N-terminal of RP-bombesin, comprising four prolines and three basic residues, is extensively different from other bombesin-like peptides. Since sequence of Pro-Arg-Pro generally plays an important role in the antimicrobial activity of proline-rich antimicrobial peptides, the componential feature of PR-bombesin indicates that it may have antimicrobial activity. In this paper, we presented the first evidence that bombesin-like peptides possess direct antimicrobial activities as some neuropeptides. It was determined by CD spectroscopy that PR-bombesin adopted a combination of random coil and beta-sheet structure, suggesting RP-bombesin is a new member of antimicrobial peptides having beta structure but without disulfide bonds. Current results also supported that PR-bombesin plays a direct defensive role besides its neuro-endocrological functions.     

iTRAQ-coupled 2-D LC-MS/MS analysis of cytoplasmic protein profile in Escherichia coli incubated with apidaecin IB

Apidaecins refer to a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. Accumulating evidence that proline-rich antimicrobial peptides are not-toxic to human and animal cells makes them potential candidates for the development of novel antibiotic drugs. However, the mechanism of action was not fully understood. In this study, antibacterial mechanism of apidaecins was investigated. iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered cytoplasmic proteins of Escherichia coli incubated with one isoform of apidaecins--apidaecin IB. The production of the chaperonin GroEL and its cofactor GroES, which together form the only essential chaperone system in E. coli cytoplasm under all growth conditions, was decreased in cells incubated with apidaecin IB. The decreasing of the GroEL-GroES chaperone team was further found to be involved in a new antibacterial mechanism of apidaecins. Our findings therefore provide important new insights into the antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.     

鲎源抗菌肽的研究及其潜在应用价值

鲎血细胞来源的抗菌肽,在鲎天然免疫中起至关重要的作用,它对外源病原菌具有抗性作用,降低了外源病原菌对鲎活体的致病性,增强了鲎的天然免疫能力。鲎源抗菌肽有一些不同于其他来源的抗菌肽的优势,对鲎源抗菌肽的研究有很重要的实用意义。概述了鲎源抗菌肽的性质、分子结构、基因序列及其制备,并对它们的潜在应用价值进行了论述。     

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.     

Insect antimicrobial peptides and their applications

Insects are one of the major sources of antimicrobial peptides/proteins (AMPs). Since observation of antimicrobial activity in the hemolymph of pupae from the giant silk moths Samia Cynthia and Hyalophora cecropia in 1974 and purification of first insect AMP (cecropin) from H. cecropia pupae in 1980, over 150 insect AMPs have been purified or identified. Most insect AMPs are small and cationic, and they show activities against bacteria and/or fungi, as well as some parasites and viruses. Insect AMPs can be classified into four families based on their structures or unique sequences: the α-helical peptides (cecropin and moricin), cysteine-rich peptides (insect defensin and drosomycin), proline-rich peptides (apidaecin, drosocin, and lebocin), and glycine-rich peptides/proteins (attacin and gloverin). Among insect AMPs, defensins, cecropins, proline-rich peptides, and attacins are common, while gloverins and moricins have been identified only in Lepidoptera. Most active AMPs are small peptides of 20–50 residues, which are generated from larger inactive precursor proteins or pro-proteins, but gloverins (~14 kDa) and attacins (~20 kDa) are large antimicrobial proteins. In this mini-review, we will discuss current knowledge and recent progress in several classes of insect AMPs, including insect defensins, cecropins, attacins, lebocins and other proline-rich peptides, gloverins, and moricins, with a focus on structural-functional relationships and their potential applications.     

Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides

In contrast to many antimicrobial peptides, members of the proline-rich group of antimicrobial peptides inactivate Gram-negative bacteria by a non-lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline-rich Bac7(1-35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA-null mutants, in fact showed resistance to several proline-rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1-35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline-rich antimicrobial peptides of eukaryotic origin.     

Bacteriocins: perspective for the development of novel anticancer drugs

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

     

Characterization of the in vivo forms of lacrimal-specific proline-rich proteins in human tear fluid

The tear film is complex and is rich in both peptides and proteins. Physiological factors have been shown to alter the balance of the protein components in the tear film, however, little is known of the precise stimuli that initiate these changes, or their nature and extent. Attention has been directed at the role of tear proteins in the protection of the external ocular surface, and their potential role in the pathogenesis of inflammatory and autoimmune diseases, but few lacrimal-specific proteins have been identified and demonstrated to offer a protective function at the ocular surface. The biological importance of proline-rich proteins is uncertain, although there is some evidence to indicate a potential antimicrobial function for these proteins in saliva. Despite the detection of mRNA for proline-rich proteins in lacrimal gland, the translated protein product has not been detected in tear fluid. In this study we investigate the presence of proline-rich proteins in the tear film. Human reflex tear fluid was examined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry directly, and following size exclusion high performance liquid chromatography. This revealed significant levels of a truncated form of lacrimal proline-rich protein, and a series of peptides derived the C-terminus of this protein. None of these had previously been identified in tear. Our study highlights the dangers inherent in proteomic strategies that assign an identity to a protein based on limited coverage of tryptic peptides.     

A peptidomics study reveals the impressive antimicrobial peptide arsenal of the wax moth Galleria mellonella

The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Dual mode of action of Bac7, a proline-rich antibacterial peptide

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.     

Analysis of the antimicrobial activities of a chemokine-derived peptide (CDAP-4) on Pseudomonas aeruginosa

Chemokines are key molecules involved in the control of leukocyte trafficking. Recently, a novel function as antimicrobial proteins has been described. CCL13 is the only member of the MCP chemokine subfamily displaying antimicrobial activity. To determine the key residues involved in its antimicrobial activity, CCL13 derived peptides were synthesized and tested against several bacterial strains, including Pseudomonas aeruginosa. One of these peptides, corresponding to the C-terminal region of CCL13 (CDAP-4) displayed good antimicrobial activity. Electron microscopy studies revealed remarkable morphological changes after CDAP-4 treatment. By computer modeling, CDAP-4 in alpha helical configuration generated a positive electrostatic potential that extended beyond the surface of the molecule. This feature is similar to other antimicrobial peptides. Altogether, these findings indicate that the antimicrobial activity was displayed by CCL13 resides to some extent at the C-terminal region. Furthermore, CDAP-4 could be considered a good antimicrobial candidate with a potential use against pathogens including P. aeruginosa.     

Purification and characterization of eight peptides from Galleria mellonella immune hemolymph

Defense peptides play a crucial role in insect innate immunity against invading pathogens. From the hemolymph of immune-challenged greater wax moth, Galleria mellonella (Gm) larvae, eight peptides were isolated and characterized. Purified Gm peptides differ considerably in amino acid sequences, isoelectric point values and antimicrobial activity spectrum. Five of them, Gm proline-rich peptide 2, Gm defensin-like peptide, Gm anionic peptides 1 and 2 and Gm apolipophoricin, were not described earlier in G. mellonella. Three others, Gm proline-rich peptide 1, Gm cecropin D-like peptide and Galleria defensin, were identical with known G. mellonella peptides. Gm proline-rich peptides 1 and 2 and Gm anionic peptide 2, had unique amino acid sequences and no homologs have been found for these peptides. Antimicrobial activity of purified peptides was tested against gram-negative and gram-positive bacteria, yeast and filamentous fungi. The most effective was Gm defensin-like peptide which inhibited fungal and sensitive bacteria growth in a concentration of 2.9 and 1.9 microM, respectively. This is the first report describing at least a part of defense peptide repertoire of G. mellonella immune hemolymph.     

Bioprospecting the American Alligator (Alligator mississippiensis) Host Defense Peptidome

Cationic antimicrobial peptides and their therapeutic potential have garnered growing interest because of the proliferation of bacterial resistance. However, the discovery of new antimicrobial peptides from animals has proven challenging due to the limitations associated with conventional biochemical purification and difficulties in predicting active peptides from genomic sequences, if known. As an example, no antimicrobial peptides have been identified from the American alligator, Alligator mississippiensis, although their serum is antimicrobial. We have developed a novel approach for the discovery of new antimicrobial peptides from these animals, one that capitalizes on their fundamental and conserved physico-chemical properties. This sample-agnostic process employs custom-made functionalized hydrogel microparticles to harvest cationic peptides from biological samples, followed by de novo sequencing of captured peptides, eliminating the need to isolate individual peptides. After evaluation of the peptide sequences using a combination of rational and web-based bioinformatic analyses, forty-five potential antimicrobial peptides were identified, and eight of these peptides were selected to be chemically synthesized and evaluated. The successful identification of multiple novel peptides, exhibiting antibacterial properties, from Alligator mississippiensis plasma demonstrates the potential of this innovative discovery process in identifying potential new host defense peptides.     

Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice

The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.     

Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications

Bacteriocins are heat-stable ribosomally synthesized antimicrobial peptides produced by various bacteria, including food-grade lactic acid bacteria (LAB). These antimicrobial peptides have huge potential as both food preservatives, and as next-generation antibiotics targeting the multiple-drug resistant pathogens. The increasing number of reports of new bacteriocins with unique properties indicates that there is still a lot to learn about this family of peptide antibiotics. In this review, we highlight our system of fast tracking the discovery of novel bacteriocins, belonging to different classes, and isolated from various sources. This system employs molecular mass analysis of supernatant from the candidate strain, coupled with a statistical analysis of their antimicrobial spectra that can even discriminate novel variants of known bacteriocins. This review also discusses current updates regarding the structural characterization, mode of antimicrobial action, and biosynthetic mechanisms of various novel bacteriocins. Future perspectives and potential applications of these novel bacteriocins are also discussed.     

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides' mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties.     

Antibiotic activity of reversed peptides of alpha-helical antimicrobial peptide,P18

P18 (KWKLFKKIPKFLHLAKKF-NH(2)), an a-helical antimicrobial peptide designed from cecropin Amagainin 2 hybrid, was known to have potent antimicrobial activity against bacteria as well as fungi without hemolytic activity. To find the peptides comparable or superior to the antimicrobial activity of P18, the two reversed peptides (Rev-1 and Rev-2) of P18 were designed and synthesized. These peptides were found to have similar antimicrobial activity against bacterial and fungal cells without hemolytic activity as compared with P18. Furthermore, a reversed peptide, Rev-2 was shown to have a two-fold higher activity in killing some bacterial cells than P18. Therefore, these results suggested that Rev-2 peptide seems to be an excellent candidate for developing novel peptide antibiotics.