两栖类动物Cathelicidins家族 抗菌肽研究进展

两栖类动物皮肤裸露和湿润的特性易于微生物的生长,它们为了抵御病原微生物的侵袭,在长期自然进化过程中形成了以抗菌肽(AMPs)为主要防御机制的免疫系统。抗菌肽广泛分布于动物、植物、微生物中,是生物用于抵御细菌、真菌、病毒和原虫等病原体侵袭的重要武器之一,在进化上是一类非常古老而有效的天然防御物质。Cathelicidins是脊椎动物特有的重要抗菌肽家族之一,除具有高效广谱的抗菌活性,还具有如抗炎、抗氧化、伤口修复、抑制组织损伤和促进血管生成等多种重要活性,因此Cathelicidins家族抗菌肽已成为抗感染多肽类新药的研发热点。本文将从两栖类动物Cathelicidins家族抗菌肽的一般特点、来源分布、生物合成与结构、生物学活性、作用机制及应用前景等几个方面,综合阐述国内外的研究动态。     

两栖动物皮肤结构及皮肤抗菌肽

两栖动物皮肤在自然进化过程中形成了防御病原微生物的三套防御系统,相应地具有特定结构。皮肤抗菌肽是其中先天性防御系统的主要组成部分。本文概述了两栖动物皮肤结构特点以及皮肤抗菌肽在国内外的最新研究进展,重点介绍了两栖动物皮肤腺体和蛙皮抗菌肽的种类、分子结构、抗菌机理、基因表达调控及cDNA编码特点以及基因工程等。以期系统认识和了解这些方面的研究与进展。     

大弹涂鱼皮肤转录组测序及抗菌肽基因分析

抗菌肽是鱼类用于抵御外界微生物入侵的天然防御多肽,也是开发新型药物的重要先导分子。大弹涂鱼(Boleophthalmus pectinirostris)是一种特殊的可以营两栖生活的鱼类,其皮肤表面具有丰富的粘液,其中所含抗菌肽对其免疫防御和适应两栖生活具有重要意义。为深入了解大弹涂鱼皮肤组织基因表达谱并从中筛选抗菌肽相关基因,采用新一代Illumina高通量测序平台对大弹涂鱼皮肤组织进行了转录组测序。利用Trinity软件从头组装,从测得的78 608 366条双端测序读长(paired-end read)共计6 GB的序列数据中获得119 848条高质量的蛋白质编码基因(unigene)。经公共数据库序列检索和比对,发现7个unigene编码的多肽与已知的鱼类5大家族的抗菌肽高度同源,即β-防御素(β-defensin)、hepcidin、NK-lysin、piscidin和肝表达抗菌肽-2(liver-expressed antimicrobial peptide-2,LEAP-2)。最后,对上述抗菌肽相关unigene开展了组织表达差异分析、序列比对及进化树分析。研究结果为进一步了解大弹涂鱼适应两栖生活的免疫防御机制和利用新鉴定的抗菌肽开发新型抗菌药物奠定了基础。     

真核生物核糖体合成的抗菌多肽

原核生物、植物、动物（包括脊椎动物和非脊椎动物）都可以产生由基因编码、核糖体合成的抗菌多肽［１］。已知的核糖体合成的抗菌多肽大部分是最近十几年中鉴定的。８０年代初,Ｓｔｅｉｎｅｒ［２］及Ｓａｌｓｔｅｄ等人［３］首次分离了昆虫的抗菌多肽ｃｅｃｒｏｐｉｎ和ｄｅｆｅｎｓｉｎｓ,从此人们开始关注这类抗菌多肽,研究了它们的遗传免疫性、寄主防御系统、膜蛋白的相互作用、蛋白的修饰和分泌等,从而将这些抗菌多肽研制开发成有应用价值的食品添加剂和药物。原核生物产生的抗菌多肽（ｎｉｓｉｎ）早已广泛应用于食品的防腐保鲜…     

中国林蛙皮肤抗菌肽抗菌的特性

从林蛙皮肤中分离到具有抗菌活性的多肽混合物——多肽FⅢ。抑菌实验表明,林蛙皮肤中小分子活性肽对革兰氏阳性细菌、革兰氏阴性细菌都具有一定的抗菌作用,并且此粗提物的抗菌活性远远高于传统食品防腐剂苯甲酸钠和山梨酸钾的抗菌活性。     

哺乳动物β-防御素研究进展

哺乳动物β-防御素是一类小分子的阳离子抗菌多肽。其具有独特的抗菌机制和广谱抗菌、抗病毒活性,同时还可作为免疫调节剂来激活与调节机体的免疫系统,在医药开发或是动植物抗病育种等方面都具有很好的应用前景。就哺乳动物β-防御素的分布、分子结构、生物学活性、作用机制及其应用等方面的研究作一综述。     

抗菌肽及抗菌肽转基因植物研究进展

抗菌肽是一类小分子多肽,在生物体内分布广泛,具有广谱的抗菌性,是生物体内天然防御系统的一部分。主要介绍了抗菌肽的性质,类型,作用机制及抗菌肽转基因植物的研究进展。     

多聚半乳糖醛酸酶抑制蛋白的结构、功能以及应用研究进展

真菌病害严重影响植物的生长发育。为了自我保护,植物进化出了许多抵御病原真菌入侵的策略,例如防御相关蛋白的产生。多聚半乳糖醛酸酶抑制蛋白（polygalacturonase-inhibiting proteins,PGIPs）是近年来研究较多的一种植物防御蛋白,它能与真菌分泌的多聚半乳糖醛酸酶（polygalacturonases,PGs）特异性结合,降低PGs水解植物细胞壁的活性并在植物体内累积能激活多种防御反应的长链寡聚半乳糖醛酸（oligogalacturonides,OGs）,从而达到抑制真菌侵染的目的。主要介绍了PGIPs的结构、功能及其抗菌机理,并综述了PGIPs在国内外转基因抗病育种中的应用研究进展。     

人防御素的抗病毒免疫作用及其研究进展

人防御素是由中性粒细胞、小肠Paneth细胞以及粘膜上皮细胞等产生的一类内源性阳离子多肽。最早因其具有广谱抗菌作用而被广泛关注。近年来研究发现,防御素对病毒也具有显著抑制作用,其抗病毒效应表现在多个方面。除了能够直接作用于病毒外,此类多肽分子还可以通过介导免疫反应来间接发挥抗病毒作用。本文就人防御素的抗病毒作用机理及其研究进展进行了综述,期望能够加强人们对防御素生物学功能的认识,并为开发相关抗病毒药物提供参考。     

研究选萃

植物用“容忍伤害”抵御昆虫进攻有一句谚语说得好——你的敌人最清楚你的弱点。这在共同进化的植物和捕食者之间表现得尤为清楚。当捕食者进化出新的进攻方法,某些植物会用独特的防御策略进行应对,乳草属植物就属于这一类。美国科学家近日研究发现,乳草属植物具有一个新的进化     

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.     

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.     

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 beta-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.     

Anti-infection peptidomics of amphibian skin

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel antimicrobial peptides. This contribution almost triples the number of currently reported amphibian antimicrobial peptides. The peptides could be organized into 30 divergent groups, including 24 novel groups. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of diversification suggest that point mutations as well as insertion, deletion, and "shuffling" of oligonucleotide sequences were responsible for the diversity. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. These diverse peptides exhibited both diverse secondary structure and "host defense" properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This has led us to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20-30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. The antimicrobial mechanisms of O. grahami peptides were investigated. They exerted their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. With respect to the development of antibiotics, these peptides provide potential new templates to explore further.     

Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI-sVIII) by cloning from a skin secretion-derived cDNA library

The defensive skin secretions of many amphibians contain a wide spectrum of biologically active compounds, particularly antimicrobial peptides that act as a first line of defence against bacterial infection. Here we describe for the first time the identification of three novel dermaseptin-related peptides (dermaseptins sVI-sVIII) whose primary structures were deduced from cDNAs cloned from a library constructed from lyophilised skin secretion of the South American hylid frog, Phyllomedusa sauvagei. The molecular masses of each were subsequently confirmed by interrogation of archived LC/MS files of fractionated skin secretion followed by automated Edman degradation sequencing. The heterogeneity of primary structures encountered in amphibian skin antimicrobial peptides may in part be explained by individual variation-a factor essential for selective functional molecular evolution and perhaps, ultimately in speciation.     

Molecular strategies in biological evolution of antimicrobial peptides

Gene-encoded antimicrobial peptides that protect the skin of hylid and ranin frogs against noxious microorganisms are processed from a unique family of precursor polypeptides with a unique pattern of conserved and variable regions opposite to that of conventional secreted peptides. Precursors belonging to this family, designated the preprodermaseptin, have a common N-terminal preproregion that is remarkably well conserved both within and between species, but a hypervariable C-terminal domain corresponding to antimicrobial peptides with very different lengths, sequences, charges and antimicrobial spectra. Each frog species has its own distinct panoply of 10-20 antimicrobial peptides so that the 5000 species of ranids and hylids may produce approximately 100,000 different peptide antibiotics. The strategy that these frogs have evolved to generate this enormous array of peptides includes repeated duplications of a 150 million years old ancestral gene, focal hypermutation of the antimicrobial peptide domain maybe involving a mutagenic DNA polymerase similar to Escherichia coli Pol V, and subsequent actions of positive (diversifying) selection. The hyperdivergence of skin antimicrobial peptides can be viewed as the successful evolution of a multi-drug defense system that provides frogs with maximum protection against rapidly changing microbial biota and minimizes the chance of microorganisms developing resistance to individual peptides. The impressive variations in the expression of frog skin antimicrobial peptides may be exploited for discovering new molecules and structural motifs targeting specific microorganisms for which the therapeutic armamentarium is scarce.     

Antimicrobial Peptide defenses in amphibian skin

One of the most urgent problems in conservation biology todayis the continuing loss of amphibian populations on a globalscale. Recent amphibian population declines in Australia, CentralAmerica, the western United States, Europe, and Africa havebeen linked to a pathogenic chytrid fungus, Batrachochytriumdendrobatidis, which infects the skin. The skin of amphibiansis critical for fluid balance, respiration, and transport ofessential ions; and the immune defense of the skin must be integratedwith these physiological responses. One of the natural defensesof the skin is production of antimicrobial peptides in granularglands. Discharge of the granular glands is initiated by stimulationof sympathetic nerves. To determine whether antimicrobial skinpeptides play a role in protection from invasive pathogens,purified antimicrobial peptides and natural peptide mixturesrecovered from the skin secretions of a number of species havebeen assayed for growth inhibition of the chytrid fungus. Thegeneral findings are that most species tested have one or moreantimicrobial peptides with potent activity against the chytridfungus, and natural mixtures of peptides are also effectiveinhibitors of chytrid growth. This supports the hypothesis thatantimicrobial peptides produced in the skin are an importantdefense against skin pathogens and may affect survival of populations.We also report on initial studies of peptide depletion usingnorepinephrine and the kinetics of peptide recovery followinginduction. Approximately 80 nmoles/g of norepinephrine is requiredto deplete peptides, and peptide stores are not fully recoveredat three weeks following this treatment. Because many specieshave defensive peptides and yet suffer chytrid-associated populationdeclines, it is likely that other factors (temperature, conditionsof hydration, "stress," or pesticides) may alter normal defensesand allow for uncontrolled infection.     

Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima

Amphibian skin secretions are rich in antimicrobial peptides acting as important components of innate defense system against invading microorganisms. A novel type of peptide, designated as maximin S, was deduced by random sequencing of 793 clones from a constructed Bombina maxima skin cDNA library. The putative primary structures of maximin S peptides can be grouped into five species, in which maximin S1 has 14 amino acid residues and the rest of maximin S peptides (S2-S5) all have 18 amino acid residues. Unlike most of the amphibian antimicrobial peptides so far identified, the newly characterized four maximin S precursors are composed of maximin S1 and different combinations of tandem repeated maximin S2-S5 linked by internal peptides. Except maximin S1, the predicted secondary structures of maximin S2-S5 show a similar amphipathic alpha-helical structure. MALDI-TOF mass spectrometry analysis of partially isolated skin secretions of the toad indicates that most of the deduced maximin S peptides are expressed. Two deduced maximin S peptides (S1, S4) were synthesized and their antimicrobial activities were tested. Maximin S4 only had an antibiotic activity against mycoplasma and had no antibacterial or antifungal activity toward tested strains. Maximin S1 had no activity under the same conditions.