抗菌肽的设计及其应用

抗菌肽(AMP)是生物体内先天免疫系统的一个组成部分,保护机体免受致病微生物的入侵.抗菌肽具有很强的广谱抗菌活性,可抑制革兰氏阳性菌、革兰氏阴性菌、真菌和病毒的生长.为克服微生物对抗生素耐药性的问题,目前阳离子抗菌肽已被考虑作为抗生素的潜在替代品.本文将阐述抗菌肽的作用机理、选择性抗菌肽的设计及其应用.     

De-Novo Design of Antimicrobial Peptides for Plant Protection

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents.     

Peptido-mimetic Approach in the Design of Syndiotactic Antimicrobial Peptides

Biocompatibility, low toxicity and high selectivity towards bacterial cells has been the hallmark of peptide-based antibiotics. The innate immune system has been employing such molecular systems against invading pathogens as a successful defense strategy. In this study, we attempt to develop topologically constrained antimicrobial peptides with syndiotactic stereochemical arrangement, by incorporating L and D amino acids successively in its amino acid sequence. Acetylated versions of the designed peptides were also examined for its influence on bactericidal potency, against Gram-positive and Gram-negative bacteria. Syndiotactic stereochemical arrangement of the polypeptide main chain mimics stereochemistry of Gramicidin, a naturally occurring antimicrobial peptides. Gramicidin is a class of penta-deca-peptides isolated from soil bacteria Bacillus brevis, but their utility as antibiotic was limited to topical use due to high levels of hemotoxicity. Activity profiles of the four de novo designed peptide variants show higher specificity towards Gram-positive bacteria than Gram-negative variants, matching earlier reports on the therapeutic potential of gramicidin as a broad spectrum antibiotic. Significantly, our hemolytic assay confirms very low (<1%) levels of toxicity for the designed peptides unlike gramicidin. Earlier reports confirm that incorporation of D amino acids effectively negates the possibility of proteolytic degradation, thus pointing to the potential utility of de novo designed peptides with diversified stereochemistry as a promising new approach in the generation of novel antibiotic peptides.     

Hydrocarbon Stapled Antimicrobial Peptides

Antimicrobial peptides are promising candidates for anti-infective pharmaceuticals. Unfortunately, because of their low proteolytic and chemical stability, their usage is generally narrowed down to topical formulations. Until now, numerous approaches to increase peptide stability have been proposed. One of them, peptide hydrocarbon stapling, a modification based on stabilizing peptide secondary structure with a side-chain covalent hydrocarbon bridge, have been successfully applied to many peptides. Moreover, constraining secondary structure of peptides have also been proven to increase their biological activity. This review article describes studies on hydrocarbon stapled antimicrobial peptides with respect to improved drug-like properties.     

贻贝抗菌肽的研究进展

贻贝中存在多种抗菌肽,根据一级结构的不同分为4种：defensin(防卫肽)、mytilin、myticin和mytimycin。抗菌肽以前体的形式存在,在缺乏感染等刺激物的情况下。成熟前体以活性方式储存在血液中,当受到感染后便参与组织反应。它们的抗感染反应与哺乳动物吞噬细胞的作用相似,不同的抗菌肽具有不同的抗菌谱。抗菌肽是机体在进化过程中非特异性免疫系统中的一种。     

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.     

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

A novel approach for boosting antimicrobial peptides through end tagging with hydrophobic oligopeptide stretches is demonstrated. Focusing on two peptides derived from kininogen, GKHKNKGKKNGKHNGWK (GKH17) and HKHGHGHGKHKNKGKKN (HKH17), tagging resulted in enhanced killing of Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and fungal Candida albicans. Microbicidal potency increased with tag length, also in plasma, and was larger for Trp and Phe stretches than for aliphatic ones. The enhanced microbicidal effects correlated to a higher degree of bacterial wall rupture. Analogously, tagging promoted peptide binding to model phospholipid membranes and liposome rupture, particularly for anionic and cholesterol-void membranes. Tagged peptides displayed low toxicity, particularly in the presence of serum, and resisted degradation by human leukocyte elastase and by staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo and in vivo in porcine S. aureus skin infection models. The generality of end tagging for facile boosting of antimicrobial peptides without the need for post-synthesis modification was also demonstrated.     

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals across the landscape is of primary importance in watershed management. Traditional economics methods of finding the lowest-cost solution in the watershed context (e.g.,^5,12,20) assume that off-site impacts can be accurately described as a proportion of on-site pollution generated. Such approaches are unlikely to be representative of the actual pollution process in a watershed, where the impacts of polluting sources are often determined by complex biophysical processes. The use of modern physically-based, spatially distributed hydrologic simulation models allows for a greater degree of realism in terms of process representation but requires a development of a simulation-optimization framework where the model becomes an integral part of optimization.Evolutionary algorithms appear to be a particularly useful optimization tool, able to deal with the combinatorial nature of a watershed simulation-optimization problem and allowing the use of the full water quality model. Evolutionary algorithms treat a particular spatial allocation of conservation practices in a watershed as a candidate solution and utilize sets (populations) of candidate solutions iteratively applying stochastic operators of selection, recombination, and mutation to find improvements with respect to the optimization objectives. The optimization objectives in this case are to minimize nonpoint-source pollution in the watershed, simultaneously minimizing the cost of conservation practices. A recent and expanding set of research is attempting to use similar methods and integrates water quality models with broadly defined evolutionary optimization methods^{3,4,9,10,13-15,17-19,22,23,25}. In this application, we demonstrate a program which follows Rabotyagov et al.''s approach and integrates a modern and commonly used SWAT water quality model⁷ with a multiobjective evolutionary algorithm SPEA2²⁶, and user-specified set of conservation practices and their costs to search for the complete tradeoff frontiers between costs of conservation practices and user-specified water quality objectives. The frontiers quantify the tradeoffs faced by the watershed managers by presenting the full range of costs associated with various water quality improvement goals. The program allows for a selection of watershed configurations achieving specified water quality improvement goals and a production of maps of optimized placement of conservation practices.     

A Crosstalk on Antimicrobial Peptides

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides (AMPs) are small sized protein molecules which play a crucial role in host inborn immune framework. AMPs with...     

Cyclic Peptides Arising by Evolutionary Parallelism via Asparaginyl-Endopeptidase-Mediated Biosynthesis

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.     

抗菌肽数据库简介

随着抗菌肽研究的不断发展,科研工作者建立了抗菌肽数据库。这些数据库使得抗菌肽的研究更加方便。就几个重要抗菌肽数据库的内容和使用方法作一简介     

Synthetic Antimicrobial Peptides: I. Antimicrobial Activity of Amphiphilic and Nonamphiphilic Cationic Peptides

Comparative antimicrobial properties of three artificial cationic synthetic antimicrobial peptides (SAMP): (RAhaR)₄AhaβA (where R is Arg, Aha is 6-aminohexanoic acid, βA is beta-alanine), (KFF)₃K and R₉F₂ with various amphiphilic properties have been studied relative to pathogenic strains of microorganisms: Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Salmonella enterica, Gram-positive bacteria Staphylococcus aureus, and pathogenic yeast fungus Candida albicans. The selectivity index (SI) values of the peptide preparations were calculated as the ratio of the 50% cytotoxic concentration (TC₅₀) towards eukaryotic host cells to the MIC₅₀ values of the testing antimicrobial peptides. The studied SAMPs appeared to be the most active against the pathogenic yeast fungus C. albicans and the bacterial strains St. aureus and P. aeruginosa. The SI values in these cases exceed 40. Some assumed molecular interactions of the studied SAMPs on the microbial cells have been considered, and possible pathways to increase their antimicrobial activity have been suggested. The proposed SAMPs can serve as a basis for the design and synthesis of new promising synthetic antimicrobial agents.     

Design and Synthesis of Heterocyclic Conjugated Peptides as Novel Antimicrobial Agents

Antimicrobial peptides have been recognized as a novel class of antibiotics and several candidates are currently in clinical trials. In the present study, new antimicrobial compounds were synthesized by coupling quinazolinone moiety with the fragments of elastin sequences VP, GVP, VGVP and GVGVP. They were evaluated for their antibacterial activity against both gram positive and gram negative bacterial strains. We are here reporting that heterocyclic conjugated tetra peptide and penta peptide showed enhanced antibacterial activity compare to the conventional antimicrobial drugs.     

Multivalent Antimicrobial Peptides as Therapeutics: Design Principles and Structural Diversities

This review highlights the design principles, progress and advantages attributed to the structural diversity associated with both natural and synthetic multivalent antimicrobial peptides (AMPs). Natural homo- or hetero-dimers of AMPs linked by intermolecular disulfide bonds existed in the animal kingdom, but the multivalency strategy has been adopted to create synthetic branched or polymeric AMPs that do not exist in nature. The multivalent strategy for the design of multivalent AMPs provides advantages to overcome the challenges faced in clinical applications of AMPs, such as: stability, efficiency, toxicity, maintenance of activity in high salt concentrations and under physiological conditions, and importantly overcoming bacterial resistance which is currently a leading health problem in the world. The multivalency strategy is valuable for moving multivalent AMPs toward clinical applications.     

抗菌肽的研究进展

抗菌肽又称抗微生物肽(antimicrobial peptide)或肽抗生素(peptide antibiotics),在动植物体内分布广泛,是天然免疫防御系统的一部分。抗菌肽不仅有广谱抗细菌能力,而且对真菌、病毒及癌细胞也有作用。对抗菌肽作用机理的研究是近来的热点之一,本文综述了此方面近来的进展,并对微生物针对抗菌肽的耐药性进行了讨论。     

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。     

新疆家蚕抗菌肽抗菌作用的超微结构观察及抗菌机理初探

为探讨基因工程表达的新疆家蚕(Bombyx mori)抗菌肽(cecropin-XJ)的抗菌机制,通过紫外分光光度法研究抗菌肽的抑菌动力学,并采用透射电镜观察抗菌肽作用于金黄色葡萄球菌(Staphylococcus aureus)后的超微结构,对抗菌肽抗菌机理进行初步探讨。结果表明,抗菌肽抑菌作用比较明显,抗菌肽的活性与作用时间有关。抗菌肽可能是通过"桶-板"模式渗透细胞膜,从而影响细胞膜的结构和功能,使细胞膜形成许多孔道,增强了金黄色葡萄球菌细胞的通透性,造成细胞内的原生质扩散,并从孔道向胞外渗漏,影响了细菌的代谢系统,从而起到抑菌、杀菌作用。抗菌肽使金黄色葡萄球菌细胞内容物大量渗漏而死亡,死亡细胞的细胞壁保持完整,表明细胞膜是抗菌肽作用的主要靶位点。     

Casein-Derived Antimicrobial Peptides Generated by Lactobacillus acidophilus DPC6026

Three peptides produced by a Lactobacillus acidophilus DPC6026 fermentation of sodium caseinate and showing antibacterial activity against pathogenic strains Enterobacter sakazakii ATCC 12868 and Escherichia coli DPC5063 were characterized. These peptides were all generated from bovine α_s1-casein and identified as IKHQGLPQE, VLNENLLR, and SDIPNPIGSENSEK. These peptides may have bioprotective applicability and potential use in milk-based formula, which has been linked to E. sakazakii infection in neonates.     

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。     

Rapid Screening of Antimicrobial Synthetic Peptides

Increasing resistance to conventional antibiotics among microorganisms is one of the leading problems of medicine nowadays. Antimicrobial peptides are compounds exhibiting both antibacterial and antifungal activities. However, it is difficult to predict whether a designed new compound would exhibit any biological activity. Moreover, purification of the peptides is one of the most time-consuming and expensive steps of the synthesis that sometimes leads to unnecessary loss of solvents and reagents. In our study we have developed a thin-layer chromatography (TLC) direct bioautography technique for rapid determination of antimicrobial activity of peptides without the necessity of high-performance liquid chromatography purification. In this assay, crude peptides were applied and separated on a TLC plate. Then, pre-prepared plates were dipped into microbial suspension and incubated under optimum conditions for bacteria and fungi as well. The activity of the tested compounds was visualized by spraying the TLC plates with a cell viability reagent, resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide). Effectiveness of this assay was compared with minimal inhibitory concentration results obtained by broth microdilution assay. Interestingly, so far such a screening method has not been applied for this group of compounds.