苦荞α-螺旋发夹抗菌肽的分子改造抑制植物真菌活性

本研究对来源于苦荞的α-螺旋发夹抗菌肽FtAMP抗真菌机制与结构之间的关系进行了研究。首先人工合成了FtAMP分子中N-端和C-端的α-螺旋(FtAMP-N和FtAMP-C）,探究两个α-螺旋究竟是哪个螺旋在起抗菌作用。然后以FtAMP为模板,α-螺旋区电荷和两亲性特征为变化要素,利用螺旋轮投影和特定氨基酸残基替换的方法,对其进行初步分子改造,并通过对多肽结构和活性比较,探讨FtAMP结构-功能的关系。研究表明,FtAMP-N和FtAMP-C都显示出良好的抗菌活性。根据螺旋轮投影方法分析螺旋的两亲性特征,并以FtAMP氨基酸序列为模板,分别表达4个FtAMP突变体(FtAMP-E12A、FtAMP-E12A/E9K、FtAMP-E12A/E9A和FtAMP-E12A/E9K/T24E）。圆二色光谱分析显示,4个多肽都可正确折叠成α-螺旋结构,在208 nm和222 nm处有典型的双负峰,表明氨基酸的改变及其表达过程中并未改变多肽的二级结构。抗真菌活性分析显示,与FtAMP相比,4种突变体对植物真菌的抑制作用均有一定增强。特别是FtAMP-E12A/E9K突变体,其抗真菌作用增强约1倍,同时诱导溶血活性并不显著,选择特异性提高近2倍。该研究也进一步表明,α-螺旋发夹抗菌肽发挥抗真菌效应主要与其螺旋结构有关,而与其抑制剂的活性位点没有关系,为该类抗菌肽结构和功能的关系提供了一定的参考。     

铰链结构对抗菌肽生物活性的影响及在分子设计中的应用研究进展

铰链结构,又称铰链区或转角,是部分抗菌肽序列中存在的一种特殊结构。但目前抗菌肽结构的研究多集中于标准的α-螺旋和β-折叠二级结构,对于铰链结构及其作用总结较少。铰链结构对抗菌肽生物活性有重要影响,主要原因是铰链结构能够提高抗菌肽的结构灵活性,促进其对细菌细胞膜的破坏作用或与胞内作用靶点的结合效率,进而提高抗菌肽的抗菌活性。同时,降低的抗菌肽结构刚性,消减了抗菌肽对真核细胞的毒性。文中结合了笔者课题组相关工作,就铰链结构特点、对抗菌肽生物活性的影响以及在抗菌肽分子设计方面的应用进行了综述,以期为新型抗菌肽的设计和开发提供参考。     

抗菌肽AD基因的合成

设计并合成了一种新型抗菌肽基因,合成的抗菌肽(cecropin)AD基因全长140个碱基对,克隆于pCR^TM2.1载体上,经DNA序列分析证实,合成的cecropin AD基因的碱基序列与设计序列完全一致.     

一类蛙源非典型结构抗菌肽cDNA的克隆以及成熟肽的预测

抗菌肽是两栖类非特异性免疫的重要组成部分,具有广谱的抗菌、抗病毒、抗肿瘤、抗寄生虫等活性,而且不易产生耐药性.抗菌肽的功能发挥与其特有的α螺旋结构密切相关,但是一些抗菌肽在水溶液中呈无规则卷曲,在类膜溶液中才转变成α螺旋,这类抗菌肽往往表现出极强的抗菌肽活性或细胞毒性,在药物开发中提供更多的利用潜能.本文从东北林蛙(Rana dybowskii)皮肤组织中,通过RT-PCR技术,克隆了一类无规则卷曲的抗菌肽,属于chensirin-2家族.19条不同的抗菌肽的cDNA序列共编码3种长度为14个氨基酸残基的成熟肽,分子量在1450.78-1460.82之间,理论等电点在9.53-9.70之间,3种抗菌肽都有两亲性和阳离子性,二级结构呈现无规卷曲.这些理化性质预示着这3种抗菌肽可能具有特殊的药用价值.     

东北林蛙皮肤中一类新抗菌肽cDNA的克隆及成熟肽的预测

为进一步研究和开发高效广谱天然抗生素的抗菌肽,本文克隆东北林蛙(Rana dybowskii)的抗菌肽基因,并预测其成熟肽的有关性质。根据蛙属抗菌肽信号肽末端序列设计简并引物,以RT-PCR技术扩增皮肤中抗菌肽的cDNA,并进行克隆测序。用生物信息学软件分析cDNA序列特点,预测成熟肽的理化性质。研究发现一种长度为28个氨基酸残基的新抗菌肽dybowsin-1,该肽具有Rana box结构;与已发现的抗菌肽仅有35%的同源性;理论等电点在9.70-10.01之间;均呈阳离子性;从第3或4个氨基酸开始到第16个氨基酸形成α-螺旋结构,极性氨基酸位于螺旋轮的一侧,非极性氨基酸位于螺旋轮的另一侧;具有N-端疏水、C-端亲水的两亲性。一个个体表达5条cDNA序列编码3种不同的dybowsin-1分子,显示出该抗菌肽表达的多样性。序列分析显示,该抗菌肽可能由多基因座位编码。     

绿僵菌素A对家蚕cecropin B和gloverin 4基因表达的影响

绿僵菌素A(DA)是由金龟子绿僵菌Metarhizium anisopliae产生的一种环缩羧肽类次生化合物,具有抗昆虫免疫作用,但是人们对其影响免疫相关基因调控的机理缺乏了解。本实验以家蚕Bm12细胞为材料,采用RNAi技术沉默相关转录因子,结合荧光定量PCR(q PCR)技术,明确DA处理或沉默TOLL和IMD信号通路相关转录因子后抗菌肽cecropin B和gloverin 4基因表达的变化。实验发现,DA能引起抗菌肽cecropin B基因表达上调和gloverin 4基因表达下调。利用特异性siRNA分别沉默转录因子BmRelish1、BmRelish2、BmRel、Bm FOXg1后,发现只有沉默转录因子BmRelish时,cecropin B和gloverin 4基因表达才下调,说明这两种抗菌肽的合成均通过IMD信号通路调控。当沉默BmRelish1或BmRel基因及DA处理联合作用时,cecropin B基因显著下调,说明在抑制cecropin B合成时,DA与转录因子BmRelish1或者BmRel之间存在密切的协同效应;同样,在促进gloverin 4合成时,DA与转录因子BmRelish2之间也存在着协同效应。     

猪源抗菌肽PMAP-36的序列设计及其结构优化的研究进展

抗生素的耐药性和动物源性食品中的药物残留问题严重威胁全球公共卫生系统。因此,开发出不易产生耐药性、抗菌活性高的新型抗菌药物迫在眉睫。抗菌肽因其分子量小、抗菌谱广、不易产生耐药性等优点受到科学家们的广泛关注,但天然抗菌肽具有抗菌活性低、溶血活性和细胞毒性等缺陷。随着抗菌肽序列和结构的不断优化,多种具有显著体内外抗菌活性且安全高效的新型抗菌药物被研发出来。猪源抗菌肽PMAP-36是从猪骨髓细胞中分离出来的一种具有典型两亲性α-螺旋结构的高阳离子抗菌肽。本文就国内外关于猪源抗菌肽PMAP-36的序列设计及其结构优化等方面的研究进展进行综述。     

东北林蛙抗菌肽dybowskin-1ST的结构预测及生物学活性分析

运用生物信息学方法进行东北林蛙抗菌肽dybowskin-1ST的进化、结构及抗原表位预测,分析其抑菌机理及结构性质,应用小鼠伤口愈合实验及体外抑菌实验进行活性验证。同时为改良亲本肽、进行新型衍生肽的研发提供理论基础。使用软件MEGA_X对dybowskin-1ST及其他蛙类抗菌肽进行同源性比对并绘制系统进化树;通过在线软件ProtParam、ProtScale、PeptideCutter、SignalP、TMHMM Server分别预测抗菌肽dybowskin-1ST的理化参数、亲/疏水性、剪切位点、信号肽及跨膜区域;分别应用 SOPMA、Jpred4及DNAstar Protean软件多重分析预测dybowskin-1ST的二级结构,利用SWISS-MODEL和I-TASSER软件进行三级结构预测。通过在线软件ABCpred和SYFPEITHI进行T/B抗原表位预测。构建小鼠伤口模型,观察dybowskin-1ST促进伤口愈合活性。应用纸片法及96孔板法,确定dybowskin-1ST的抑菌活性。抗菌肽dybowskin-1ST含有59个氨基酸,其中亮氨酸占16.9%,分子式为C318H510N80O93S2,理论等电点为5.10,电荷量为?2。抗菌肽dybowskin-1ST与东北林蛙抗菌肽dybowskin-1CDYa亲缘较近。三种方法二级结构预测结果相似,dybowskin-1ST中α-螺旋、延伸链、β-转角、无规卷曲,所占比例分别为44.07%、16.95%、3.39%、35.39%。三级结构预测中显示该抗菌肽大部分为α-螺旋结构,抗菌肽dybowskin-1ST总体预测为亲水性蛋白,具有信号肽序列。亚细胞定位分析显示,其分泌线粒体靶向肽的可能性为0.944。该蛋白属于膜外蛋白,无跨膜结构区,有7个磷酸化位点,3个T细胞抗原表位和8个B细胞抗原表位。dybowskin-1ST具有促进伤口愈合的作用,能够有效抑制大肠杆菌和金黄色葡萄球菌活性,但对真菌及耐药菌的抑菌活性有限。dybowskin-1ST结构中虽富含α-螺旋,但验证实验表明其抑菌能力仍有待加强,原因可能是由于其带负电荷且为亲水性蛋白,以提高正电荷数及改变疏水性为基本思路进行氨基酸改造可获得活性升级的衍生肽。     

利用精氨酸和缬氨酸设计新型α-螺旋抗菌肽

摘要：【目的】抗菌肽是生命体的自身免疫系统的重要组成部分。其中两性的α-螺旋抗菌肽在抗菌肽家族中又占有重要的地位,发挥着重要的作用。为了得到具有更高抗菌活性同时具有很低细胞毒性的抗菌肽,根据α-螺旋二级结构衍生出来的螺旋轮模型,设计了一条在疏水一侧含有8个缬氨酸和亲水一侧含有5个精氨酸的新型16残基抗菌肽。【方法】测定了设计得到的新型抗菌肽的最小抑菌浓度、对于红细胞和哺乳动物肾细胞的细胞毒性以及杀菌动力学。 【结果】抗菌活性检测表明,新型抗菌肽VGR16显示了强并快速的杀菌作用,其最小抑菌浓度在16－64     

利用亮氨酸和赖氨酸设计新型α-螺旋抗菌肽

【目的】根据螺旋轮模型设计以亮氨酸(L)为疏水面,赖氨酸(K)为亲水面的新型α-螺旋抗菌肽LK,并对该抗菌肽的生物学活性进行检测。【方法】利用圆二色光谱分析LK的二级结构,同时,评价LK的抑菌活性、稳定性和细胞选择性。【结果】在模拟细胞膜的环境中LK呈α-螺旋型结构。LK对多种革兰氏阳性菌和革兰氏阴性菌有很强的抑菌活性,最小抑菌浓度(MIC)在2-4μmol/L之间。LK具有很强的酸碱盐稳定性。肽浓度为2-4μmol/L时,LK表现出较低的溶血活性和细胞毒性。【结论】根据螺旋轮模型结构,以疏水性的L和正电荷性的K设计的新型抗菌肽LK具有较高的细胞选择性及稳定性,具有替代抗生素的发展潜力。     

Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.     

High-throughput generation of small antibacterial peptides with improved activity

Cationic antimicrobial peptides are able to kill a broad variety of Gram-negative and Gram positive bacteria and thus are good candidates for a new generation of antibiotics to treat multidrug-resistant bacteria. Here we describe a high-throughput method to screen large numbers of peptides for improved antimicrobial activity. The method relies on peptide synthesis on a cellulose support and a Pseudomonas aeruginosa strain that constitutively expresses bacterial luciferase. A complete substitution library of 12-amino-acid peptides based on a linearized variant (RLARIVVIRVAR-NH(2)) of the bovine peptide bactenecin was screened and used to determine which substitutions at each position of the peptide chain improved activity. By combining the most favorable substitutions, we designed optimized 12-mer peptides showing broad spectrum activities with minimal inhibitory concentrations (MIC) as low as 0.5 microg/ml against Escherichia coli. Similarly, we generated an 8-mer substituted peptide that showed broad spectrum activity, with an MIC of 2 microg/ml, against E. coli and Staphylococcus aureus.     

Isolation and functional analysis of a 24-residue linear alpha-helical antimicrobial peptide from Korean blackish cicada, Cryptotympana dubia (Homoptera)

A new antimicrobial peptide, cryptonin, was isolated and characterized from the adult Korean blackish cicada, Cryptotympana dubia. It consists of 24 amino acid residues and has a molecular weight of 2,704 Da on mass spectroscopy. The predicted alpha-helical structure analysis and increased helix percent in 40% trifloroethanol of cryptonin suggests that it belongs to the typical linear alpha-helix forming peptide. Binding of the biotin-labeled cryptonin at the surface of E. coli cells and increased influx of propidium iodide in E. coli after cryptonin treatment indicates that it kills microbial cells by binding bacterial cell surfaces and disrupting the cell permeability. Cryptonin showed strong antibacterial (MIC 1.56-25 microg/ml) and antifungal (MIC 3.12-50 microg/ml) activities against tested bacteria and fungi including two antibiotic-resistant bacterial strains; methicilin-resistant S. aureus and vancomycin-resistant Enterococci (MIC 25 microg/ml, each).     

Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens

Peptides derived from the N-terminal domain that comprises an amphipathic alpha-helix in human lactoferrin (LFh 18-31 and LFh 20-38) and bovine lactoferrin (LFb 17-30 and LFb 19-37) were chemically synthesised. Since many positively charged amphipathic alpha-helices contain antimicrobial activity, the peptides were tested for their antimicrobial activity against various oral pathogens. Both peptides from bovine lactoferrin had more potent antimicrobial activities than the human equivalents. Peptide LFb 17-30, containing the largest number of positively charged amino acids, showed the highest antimicrobial activity to both Gram-positive and Gram-negative bacteria. Since native lactoferrin molecules had no killing activity, release of these peptides from the native protein should be investigated to explore the use in oral care products.     

Direct comparison of membrane interactions of model peptides composed of only Leu and Lys residues

We compared the properties of two peptides of identical size and amino acid composition, Ac-(LKKL)(5)-NHEt and Ac-(KL)(10)-NHEt. Both are amphipathic, but only Ac-(LKKL)(5)-NHEt is a potent promoter of negative curvature. CD studies performed in the presence of lipids confirmed that under these conditions Ac-(LKKL)(5)-NHEt forms an alpha-helix, and Ac-(KL)(10)-NHEt adopts a beta structure. We studied their binding affinity by centrifugation and isothermal titration calorimetry techniques. The Ac-(LKKL)(5)-NHEt bound to zwitterionic and anionic liposomes, while Ac-(KL)(10)-NHEt interacted mainly with anionic liposomes. Ac-(LKKL)(5)-NHEt was more lytic than Ac-(KL)(10)-NHEt for zwitterionic palmitoyloleoylphosphatidylcholine (POPC) liposomes, and for liposomes composed of lipids extracted from either sheep or human erythrocytes (RBC). Both peptides had similar lytic and lipid mixing activities for liposomes containing anionic lipids. Both peptides were highly hemolytic, with Ac-(LKKL)(5)-NHEt active against sheep RBC and Ac-(KL)(10)-NHEt more active against human RBC. From their respective minimal effective concentrations (MECs) as antimicrobial agents, we judged Ac-(KL)(10)-NHEt to be 2 to 5-fold more potent than Ac-(LKKL)(5)-NHEt in media that contained physiological concentrations of NaCl. Notwithstanding, both peptides had MECs <1 microg/mL for Escherichia coli and Pseudomonas aeruginosa and <4 microg/mL for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Although selectivity of antimicrobial peptides for bacterial membranes may result, in part, from the preferential display of anionic residues in these membranes, inability to interact with or bind to zwitterionic phospholipids offers no guarantee that the peptide will lack appreciable cytotoxicity for host cells.     

Histatin and lactoferrin derived peptides: antimicrobial properties and effects on mammalian cells

In order to analyze the clinical potential of two antimicrobial peptides, human lactoferrin 1-11 (hLF1-11) and synthetic histatin analogue Dhvar-5, we measured the killing effect on bacteria, and the potential toxicity on erythrocytes and bone cells. The antimicrobial activity was determined in a killing assay on six strains, including methicillin resistant Staphylococcus Aureus. The effect on human erythrocytes and MC3T3 mouse bone cells was measured with a hemolysis assay and a viability assay, respectively. Both hLF1-11 and Dhvar-5 dose-dependently killed all bacterial strains, starting at concentrations of 6 μg/mL. hLF1-11 had no effect on mammalian cells at concentrations up to 400 μg/mL, but Dhvar-5 induced significant hemolysis (37% at 200 μg/mL) and bone cell death (70% at 400 μg/mL). This indicates that both peptides are able to kill various resistant and non-resistant bacteria, but Dhvar-5 may exert a cytotoxic effect on host cells at higher concentrations.     

Antibacterial activities of poly(amidoamine) dendrimers terminated with amino and poly(ethylene glycol) groups

Poly(amidoamine) (PAMAM) dendrimer derivatives have been investigated for their biological applications, especially for delivery of drugs, including antimicrobial drugs to eukaryotic cells, but their effects on bacterial cells are largely unexplored. Herein we report that amino-terminated PAMAM dendrimers are highly toxic to the common Gram-negative pathogen Pseudomonas aeruginosa. The concentration that kills 50% of the bacteria (EC50) was in the range of approximately 0.9-1.5 microg/mL for the generation 5, amino-terminated dendrimers with or without partial (43%) coating of poly(ethylene glycol) (PEG). These EC50 values were lower than that ( approximately 1.9-2.8 microg/mL) for LL-37, a potent antimicrobial peptide expressed in a variety of epithelia. On the contrary, the dendrimers were far less toxic (EC50 > 21 microg/mL) to the Gram-positive pathogen Staphylococcus aureus than LL-37 (EC50 = approximately 1.9 microg/mL). In agreement with the previous studies on other cell types, the dendrimers were not cytotoxic to human corneal epithelial cells at the concentrations that were toxic to P. aeruginosa. Our findings indicate that amino-terminated PAMAM dendrimers and their partially PEG-coated derivatives possess attractive antimicrobial properties, particularly against Gram-negative bacteria, thus expanding the potential biological application of the dendrimers.     

Antimicrobial effects of H4-(86-100), histogranin and related compounds--possible involvement of DNA gyrase

Histone-derived antimicrobial peptides have been identified in various organisms from plants to humans. The rat histone H4 mRNA variants, H4-v.1 and rat histogranin (HNr) mRNAs, were recently reported to be involved in the synthesis of H4-(86-100) and its related peptide HNr, respectively. Herein, the two peptides were investigated for putative antimicrobial activity and found to inhibit growth of gram-negative (Escherichia coli, Pseudomonas aeruginosa) and gram-positive (Bacillus subtilis, Staphylococcus aureus) bacteria. Their inhibitory potencies in E. coli (LD(50): 3.48 and 4.34 microg x mL(-1)) are comparable to that of the antimicrobial peptide LL-37 (LD(50): 4.10 microg x mL(-1)). The antimicrobial activities of H4-(86-100) and HNr depend upon the integrity of the molecules, as precursors [H4-(84-102), pro-HNr] and fragments [bovine histogranin (HNb)-(1-13), HNb-(3-13), H4-(89-102) or OGP] are at least five times less potent than the parent peptides. Among various HN-like compounds, cyclo-(-Gly-pCl-Phe-Tyr-D-Arg) (compound 3) and N-5-guanidino pentanamide-(2R)-yl-2-N-(p-hydroxyphenylacetyl)-4-(p-chlorobenzoyl)-phenylene diamine (compound 8) display antimicrobial activities comparable to that of HNr. Interestingly, the antimicrobial activities of H4-(86-100), HNr and compound 3, like those of quinolone antibiotics acting as DNA gyrase poisons, are potentiated by ATP (1 mM) and coumermycin A1 (a DNA gyrase-linked ATPase inhibitor) and blocked by 2,4-dinitrophenol (DNP, an uncoupler of oxidative phosphorylation) and fluoroacetic acid (a metabolic poison). Finally, in vitro experiments indicate that H4-(86-100), HNr, compound 3 and compound 8, but not HNb-(1-13) or HNb-(3-13), inhibit DNA gyrase-mediated supercoiling of pBR322 DNA. These data indicate that the naturally occurring H4-(86-100) and HNr display antimicrobial effects that involve a modulation of ATP-dependent DNA gyrase.     

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.     

Comparative activities of cecropin A, melittin, and cecropin A-melittin peptide CA(1-7)M(2-9)NH2 against multidrug-resistant nosocomial isolates of Acinetobacter baumannii

The in vitro activity of three polycationic peptides, cecropin A, melittin, and cecropin A-melittin hybrid peptide CA(1-7)M(2-9)NH2, alone and in combination with various clinically used antimicrobial agents, was investigated against 32 nosocomial isolates of Acinetobacter baumannii. Antimicrobial activities were measured by MIC, MBC and bacterial killing assay. The peptides demonstrated different ranges of inhibitory values: overall, the organisms were more susceptible to CA(1-7)M(2-9)NH2 (MIC range, 0.25-16 mg/l) than to cecropin A (0.50-32 mg/l) and melittin (0.50-32 mg/l). Synergy was observed when CA(1-7)M(2-9)NH2 and melittin were combined with beta-lactam antibiotics.