Amphipathic, alpha-helical antimicrobial peptides

Gene-encoded antimicrobial peptides are an important component of host defense in animals ranging from insects to mammals. They do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. They are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. A particularly abundant and widespread class of antimicrobial peptides are those with amphipathic, alpha-helical domains. Due to their relatively small size and synthetic accessibility, these peptides have been extensively studied and have generated a substantial amount of structure-activity relationship (SAR) data. In this review, alpha-helical antimicrobial peptides are considered from the point of view of six interrelated structural and physicochemical parameters that modulate their activity and specificity: sequence, size, structuring, charge, amphipathicity, and hydrophobicity. It begins by providing an overview of how these vary in peptides from different natural sources. It then analyzes how they relate to the currently accepted model for the mode of action of alpha-helical peptides, and discusses what the numerous SAR studies that have been carried out on these compounds and their analogues can tell us. A comparative analysis of the many alpha-helical, antimicrobial peptide sequences that are now available then provides further information on how these parameters are distributed and interrelated. Finally, the systematic variation of parameters in short model peptides is used to throw light on their role in antimicrobial potency and specificity. The review concludes with some considerations on the potentials and limitations for the development of alpha-helical, antimicrobial peptides as antiinfective agents.     

Lantibiotics, class I bacteriocins from the genus Bacillus

Antimicrobial peptides exhibit high levels of antimicrobial activity against a broad range of spoilage and pathogenic microorganisms. Compared with bacteriocins produced by lactic acid bacteria, antimicrobial peptides from the genus Bacillus have been relatively less recognized despite their broad antimicrobial spectra. These peptides can be classified into two different groups based on whether they are ribosomally (bacteriocins) or nonribosomally (polymyxins and iturins) synthesized. Because of their broad spectra and high activity, antimicrobial peptides from Bacillus spp. may have great potential for applications in the food, agricultural, and pharmaceutical industries to prevent or control spoilage and pathogenic microorganisms. In this review, we introduce ribosomally synthesized antimicrobial peptides, the lantibiotic bacteriocins produced by members of Bacillus. In addition, the biosynthesis, genetic organization, mode of action, and regulation of subtilin, a well-investigated lantibiotic from Bacillus subtilis, are discussed.     

Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications

Ribosomally synthesized peptides with antimicrobial properties (antimicrobial peptides-AMPs) are produced by eukaryotes and prokaryotes and represent crucial components of their defense systems against microorganisms. Although they differ in structure, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of them attack their target cells by permeabilizing the cell membrane. They can be roughly categorized into those that have a high content of a certain amino acid, most often proline, those that contain intramolecular disulfide bridges, and those with an amphiphilic region in their molecule if they assume an alpha-helical structure. Most of the known ribosomally synthesized peptides with antimicrobial functions have been identified and studied during the last 20 years. As a result of these studies, new knowledge has been acquired into biology and biochemistry. It has become evident that these peptides may be developed into useful antimicrobial additives and drugs. The use of two-peptide antimicrobial peptides as replacement for clinical antibiotics is promising, though their applications in preservation of foods (safe and effective for use in meat, vegetables, and dairy products), in veterinary medicine, and in dentistry are more immediate. This review focuses on the current status of some of the main types of ribosomally synthesized AMPs produced by eucaryotes and procaryotes and discusses the novel antimicrobial functions, new developments, e.g. heterologous production of bacteriocins by lactic acid bacteria, or construction of multibacteriocinogenic strains, novel applications related to these peptides, and future research paradigms.     

Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action

Ribosomally synthesized peptides with antimicrobial activity are produced by prokaryotes, plants, and a wide variety of animals, both vertebrates and invertebrates. These peptides represent an important defense against micro-organisms. Although the peptides differ greatly in primary structures, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of these peptides kill their target cells by permeabilizing the cell membrane. Moreover, many of these peptides may roughly be placed into one of three groups: (1) those that have a high content of one (or two) amino acid(s), often proline, (2) those that contain intramolecular disulfide bonds, often stabilizing a predominantly β-sheet structure, and (3) those with amphiphilic regions if they assume an α-helical structure. Most known ribosomally synthesized antimicrobial peptides have been identified and characterized during the past 15 years. As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as innate immunity, membrane-protein interactions, and protein modification and secretion. Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives and drugs. This review presents a broad overview of the main types of ribosomally synthesized antimicrobial peptides produced by eukaryotes and prokaryotes. Received: 30 August 1996 / Accepted: 26 November 1996     

Structures of cyclic, antimicrobial peptides in a membrane-mimicking environment define requirements for activity.

New antimicrobial compounds are of major importance because of the growing problem of bacterial resistance. In this context, antimicrobial peptides have received a lot of attention. Their mechanism of action, however, is often obscure. Here, the structures of two cyclic, antimicrobial peptides from the family of arginine- and tryptophan-rich peptides determined in a membrane-mimicking environment are described. The sequence of the peptides has been obtained from a cyclic parent peptide by scrambling the amino acids. While the activity of the peptides is similar to that of the parent peptide, the structures are not. The peptides do, however, all adopt an amphiphilic structure. A comparison between the structures helps to define the requirements for the activity of these peptides.     

抗菌肽改良设计及抗炎作用的研究进展

抗菌肽因其具有广谱抗菌活性、不容易引起抵抗性,被认为是先天免疫系统对抗微生物感染的多功能工具。然而,天然抗菌肽存在抗菌活性低、稳定性低、溶血性高等问题,使其较难应用于临床,所以研究人员对抗菌肽进行改良设计以期获得更高抗菌活性、更低溶血活性的新型抗菌肽。另外,天然抗菌肽作为一类免疫效应因子而被发现,其表现出的抑菌、免疫调节、内毒素中和等作用,使得研究人员对抗菌肽在抗炎作用的研究表现出极大的兴趣。就抗菌肽的药物设计方法及抗炎作用机制进行综述。     

Diversity of antimicrobial peptides and their mechanisms of action

Antimicrobial peptides encompass a wide variety of structural motifs. Many peptides have alpha-helical structures. The majority of these peptides are cationic and amphipathic but there are also hydrophobic alpha-helical peptides which possess antimicrobial activity. In addition, some beta-sheet peptides have antimicrobial activity and even antimicrobial alpha-helical peptides which have been modified to possess a beta-structure retain part of their antimicrobial activity. There are also antimicrobial peptides which are rich in a certain specific amino acid such as Trp or His. In addition, antimicrobial peptides exist with thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure which allows them to bind to the membrane interface. Indeed, most antimicrobial peptides interact with membranes and may be cytotoxic as a result of disturbance of the bacterial inner or outer membranes. Alternatively, a necessary but not sufficient property of these peptides may be to be able to pass through the membrane to reach a target inside the cell. The interaction of these peptides with biological membranes is not just a function of the peptide but is also modulated by the lipid components of the membrane. It is not likely that this diverse group of peptides has a single mechanism of action, but interaction of the peptides with membranes is an important requirement for most, if not all, antimicrobial peptides.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

We recently reported the primary structures, antimicrobial activities and cDNA precursors of nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorranaishikawae. Their cDNA clones revealed a highly conserved approximately 60 bp region upstream of the start codon. This conserved region was used in the “shotgun” cDNA cloning method to reveal additional cDNAs encoding novel antimicrobial peptides of O.ishikawae. After sequencing 344 clones, we identified novel 13 cDNAs encoding dermal peptides in addition to the previously identified nine antimicrobial peptides. These 13 unique cDNAs encoded precursor proteins each containing a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg/Lys processing site and a dermal peptide at the C-terminus. The dermal peptides were members of the palustrin-2 (two peptides; termed palustrin-2ISc and palustrin-2ISd), nigrocin-2 (one peptide; nigrocin-2ISc), brevinin-1 (one peptide; brevinin-1ISa), odorranain-M (one peptide; odorranain-MISa) and entirely novel peptides (eight peptides; ishikawain-1-8). Although palustrin-2ISd and odorranain-MISa had few antimicrobial activities, palustrin-2ISc and nigrocin-2ISc possessed a broad-spectrum of growth inhibition against bacteria. Brevinin-1ISa had the most potent antimicrobial activities against the Gram-positive bacteria and the fungus but not the Gram-negative bacterium, Escherichiacoli. However, eight novel peptides showed no growth inhibition against these microorganisms.     

Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications

The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635) was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring.     

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.     

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.     

蜘蛛抗菌肽研究进展

蜘蛛活性多肽研究主要集中于蜘蛛毒液中作用于离子通道的神经毒素多肽。但近年来,一些蜘蛛抗菌肽不断被分离纯化,其结构和抗菌活性也被广泛深入研究,这将成为蜘蛛活性多肽研究领域的一个新热点。在蜘蛛毒液和血液中,存在不同种类的抗菌肽,其多肽长度、结构、抗菌作用各不相同。而且,有些抗菌肽甚至具有抗肿瘤作用。概述了蜘蛛抗菌肽在结构和功能方面的研究进展。     

There are abundant antimicrobial peptides in brains of two kinds of Bombina toads

It is well-known that there is a large amount of antimicrobial peptides in amphibian skins but few antimicrobial peptides are found in amphibian brains. Twenty-two and four antimicrobial peptides were purified and characterized from the brain homogenate of Bombina maxima and B. microdeladigitora, respectively. One hundred fifty-eight cDNA clones encoding 79 antimicrobial peptides were isolated from brain cDNA libraries of B. maxima and B. microdeladigitora. These antimicrobial peptides belong to two peptide groups (maximin and maximin-H). Twenty of them are identical to previously reported antimicrobial peptides (maximin 1-8, 10, 11, maximin H1, 3-5, 7, 9, 10, 12, 15, 16) from B. maxima skin secretions. Fifty-nine of them are novel antimicrobial peptides. Some of these antimicrobial peptides showed strong antimicrobial activities against tested microorganism strains including Gram-positive and -negative bacteria and fungi. The current diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal brains. The extreme diversity may give rise to interest to prospect the actual functions of antimicrobial peptides in amphibian brains.     

Antibacterial and hemolytic activity of the skin of the terrestrial salamander, Plethodon cinereus

As resistance increases against fungal antibiotics, antimicrobial peptides are receiving attention as possible replacements. The dermal glands of frogs secrete, among other things, antimicrobial peptides. As part of the innate immune system, stressors may affect the production of antimicrobial peptides by dermal glands. The dermal secretions of some salamanders have been examined for their toxic secretions, but little attention has been given to salamander antimicrobial peptides. This study examines the skin from the tail region for the production of antimicrobial peptides in the terrestrial salamander, Plethodon cinereus. Fractions of tail extracts were isolated using cation-exchange chromatography and reverse-phase HPLC. An HPLC fraction eluting at 15.75 min (HPLC run: 30 min, 30-80% acetonitrile/water gradient, Aquapore RP-300 C18 column) showed activity against Staphylococcus aureus but not against Escherichia coli. The antibacterial activity gradually increased over a 4-hr incubation time up to about 85% inhibition of bacterial growth. Lysis of guinea pig red blood cells also increased gradually over a 1-hr time period. J. Exp. Zool. 287:340-345, 2000.     

Antimicrobials,Stress and Mutagenesis

Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs) mostly target the cell wall, a microbial ‘Achilles heel’, it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient ‘weapons’ of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs) do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the ‘Achilles heel’ has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.     

Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa

The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions. We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides. This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation. Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated. Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides. The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers. Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system. The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P. aeruginosa infections.     

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.     

Antimicrobial Peptides from Plants

Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell layer lining the organ, which is consistent with a role for the antimicrobial peptides in constitutive host defense against microbial invaders attacking from the outside. Thionins are predominantly located intracellularly but are also found in the extracellular space, whereas most plant defensins and lipid transfer proteins are deposited exclusively in the extracellular space. In a number of plant species, a strong induction of genes expressing either thionins, plant defensins, or lipid transfer proteins has been observed on infection of the leaves by microbial pathogens. Hence, antimicrobial peptides can also take part in the inducible defense response of plants. Constitutive expression in transgenic plants of heterologous antimicrobial peptide genes has been achieved, which in some cases has led to enhanced resistance to particular microbial plant pathogens.     

抗菌肽在大肠杆菌中的融合表达

抗菌肽作为新一代抗生素的潜在应用价值使其备受关注,大量高纯度的抗菌肽是开展基础及临床实验的关键。天然来源的抗菌肽资源有限、纯化困难,化学合成抗菌肽成本高、活性不稳定,因此通过基因重组表达得到大量抗菌肽是低成本、高效益的方法。目前采用大肠杆菌表达系统获得抗菌肽已成为研究者的首选,通常以形成融合蛋白的方式表达,这不仅可避免抗菌肽对宿主的杀伤作用,也保护了抗菌肽免受蛋白酶降解。文章结合课题组的研究工作,综述了近年来抗菌肽在大肠杆菌中表达的融合载体、融合蛋白的裂解方法及表达条件优化的研究进展。