Synthesis and Antibacterial Activity of Analogues of the N-Terminal Fragment of the Sarcotoxin IA Antimicrobial Peptide

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1–18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.     

粉纹夜蛾离体细胞抗菌肽的抗菌谱测定

用热灭活的大肠杆菌DHSQ诱导粉纹夜蛾(Trichoplusia ni)离体细胞产生抗菌肽,用三氯乙酸沉淀法提取出该活性物质,采用琼脂糖孔穴扩散法和生长抑制测定法测定其抗菌谱,发现该抗菌物质具有较广的抗微生物活性,其中特别是对革兰氏阴性菌中的沙门氏茵和大肠杆茵,酵母菌中的白色念珠菌,植物病源真茵中的花生白绢病茵和小麦赤霉病茵具有较强的抑菌活性,从而表明该物质是一种既抗细菌,又抗真菌的抗微生物肽。     

Immobilization of Escherichia coli Cells by Use of the Antimicrobial Peptide Cecropin P1

An immobilization scheme for bacterial cells is described, in which the antimicrobial peptide cecropin P1 was used to trap Escherichia coli K-12 and O157:H7 cells on microtiter plate well surfaces. Cecropin P1 was covalently attached to the well surfaces, and E. coli cells were allowed to bind to the peptide-coated surface. The immobilized cells were detected colorimetrically with an anti-E. coli antibody-horseradish peroxidase conjugate. Binding curves were obtained in which the signal intensities were dependent upon the cell concentration and upon the amount of peptide attached to the well surface. After normalization for the amount of peptide coupled to the surface and the relative binding affinity of the antibody for each strain, the binding data were compared, which indicated that there was a strong preference for E. coli O157:H7 over E. coli K-12. The cells could be immobilized reproducibly at pH values ranging from 5 to 10 and at ionic strengths up to 0.50 M.     

天蚕素A-爪蟾素2杂合肽P18对MDA-MB-435s乳腺癌细胞的作用研究

目的 研究天蚕素A-爪蟾素2杂合肽P18对体外培养的人类乳腺癌细胞株MDA-MB-435s的活性作用.方法 采用MTT法检测P18对体外培养的MDA-MB-435s细胞增殖的影响;使用Live/Dead Viability/Cytotoxicity试剂盒对细胞进行染色,并在荧光显微镜下观察细胞的存活状态;通过DiBAC_4(3)染色,以荧光分光光度计检测给肽时细胞膜的膜电位变化;在透射电子显微镜下观察P18作用24 h后细胞超微结构的变化.结果 MTT试验表明P18对MDA-MB-435s细胞的增殖具有浓度依赖性的抑制作用;荧光显微镜下可观察到P18可以使MDA-MB-435s细胞膜破损,细胞死亡;P18作用时,荧光分光光度计检测到MDA-MB-435s的细胞膜发生了去极化现象;透射电子显微镜下可见大量细胞崩解坏死.结论 杂合肽P18能够引起MDA-MB-435s细胞膜的通透性改变而导致细胞坏死.     

家蝇幼虫抗菌肽的超声诱导及分离纯化和活性研究

目的:分离纯化家蝇(Musca domestica)幼虫免疫血淋巴中的抗菌肽,研究抗菌肽的性质和抑菌特性.为进一步研究家蝇幼虫抗菌肽的产生及应用提供实验基础.方法:通过超声处理诱导家蝇幼虫产生免疫血淋巴,经沸水浴热变性,结合高速离心,两步CM-Sepharose离子交换层析,冷冻干燥浓缩等步骤,分离得到一种具抗菌活性的蛋白质.用Tricine-SDS-PAGE鉴定抗菌肽的纯度和性质,抑菌试验分析抗菌肽的抑菌特性.结果:300W,50Hz超声处理60s能够诱导家蝇幼虫产生抗菌肽,经分离纯化后Tricine-SDS-PAGE凝胶电泳结果显示为一条带,相对分子量大约为5.8kD.纯化蛋白对阴沟肠杆菌,绿脓杆菌等G-杆菌的抑菌活性较强,而对金黄色葡萄球菌效果不显著.结论:诱导和纯化了一种家蝇幼虫抗菌肽,为此类活性物质的分离纯化提供了有效的方式.     

Antibacterial,Cytotoxicity and Mechanism of the Antimicrobial Peptide KR-32 in Weaning Piglets

International Journal of Peptide Research and Therapeutics - Diarrhea is a serious issue among livestock, and pathogens and viral infections are the main causes of diarrhea, especially in weaned...     

新疆家蚕抗菌肽在毕赤酵母中表达及活性研究

目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性.方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽(Cecropin-XJ)基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoR Ⅰ及Not Ⅰ双酶切,构建重组表达质粒pPIC9K-(Cecropin-XJ),酶切及测序正确后,电转化到毕赤酵母GS115,对分泌表达的重组蛋白进行活性检测.结果:PCR扩增获得192 bp Cecropin-XJ,成功构建pPIC9K-Cecropin-XJ,优化诱导条件证明在pH 6的BMMY培养液中,0.5%甲醇诱导约48h后,获得的表达产物活性较强,对多种革兰氏阴性菌和阳性菌具有抗菌活性,在100℃条件下,其活性可维持100min以上.结论:新疆家蚕抗菌肽在毕赤酵母中分泌表达,为大规模发酵生产奠定了基础.     

Impact of Lauric Arginate Application Form on its Antimicrobial Activity in Meat Emulsions

Lauric arginate (LAE) is a food-grade cationic surfactant that is highly active against a wide range of food pathogens (Listeria monocytogenes, Salmonella, and Escherichia coli) and food spoilers (Lactobacilli, yeast, and molds). The antimicrobial efficacy of LAE in compositionally complex environments is likely to be negatively impacted by its interactions with food ingredients. Therefore, we investigated different application systems of LAE and their impact on its antimicrobial efficacy when added to “Lyoner style” sausages. LAE was applied as a powder, aqueous solution, in oil-in-water emulsions with different droplet sizes, and as solid lipid particles (SLP) with different droplet sizes. Structures of the systems were identified by optical microscopy, differential scanning calorimetry (DSC) and static light scattering. A recontamination on the surface of sliced sausages was simulated using Listeria innocua as the target organism (2 log colony forming units (CFU)/slice), and the antimicrobial impact of 1,000, 1,500 and 2,000 μg/g applied LAE in the sausage was examined by growth curves. A modeling of the CFU-time relationship was carried out to provide a better evaluation of the antimicrobial activity of LAE. Finally, we carried out an isothermal titration calorimetry (ITC) analysis to simulate the interactions between LAE and proteins in the sausage matrix. Results revealed that the application systems differed in their surface area and, therefore, showed different antimicrobial activities when incorporated into sausage. The study demonstrated that the SLP and emulsions as LAE application systems increased the antimicrobial activity against microbial growth on the surface of sliced “Lyoner style” sausages.     

Factors Affecting the Antimicrobial Activity of Vitamin K5

Pure cultures of Escherichia coli, Bacillus subtilis, Proteus vulgaris, Staphylococcus aureus, and Pseudomonas fluorescens were used in this investigation. The bactericidal concentrations of vitamin K(5) required for E. coli, B. subtilis, P. vulgaris, S. aureus, and P. fluorescens; the effect of an absence of oxygen; the effect of contact time with E. coli and S. aureus; and the effect of initial counts per milliliter of E. coli were studied. The bactericidal concentrations ranged from 60 ppm of K(5) for S. aureus to 220 ppm for E. coli, with an initial count of 160,000 to 200,000 cells per milliliter and a contact time of 12 hr in nutrient broth. The gram-positive bacteria tested were more susceptible to the antimicrobial activity of vitamin K(5) than the gram-negative bacteria. In the studies conducted under nitrogen atmosphere, the per cent inhibition showed an inverse relationship to the bactericidal concentrations required for complete inhibition in studies conducted under air atmosphere. This finding suggested that there might be different factors responsible for inhibition depending on the species of bacteria being tested, and it also might help explain the difference in concentrations necessary for inhibition. Cells of E. coli and S. aureus were not inhibited immediately on coming into contact with vitamin K(5); 50% inhibition occurred after 25 and 32 min, respectively. A rapid inhibition rate was maintained until approximately 90% inhibition occurred, after whch a rapid decrease in the rate was noted.     

家蝇抗菌肽Defensin在毕赤酵母中的表达及活性鉴定

拟通过酵母系统表达家蝇抗菌肽Defensin基因,并初步鉴定其抗菌活性。采用限制性内切酶SacⅠ将分泌型重组表达质粒pPIC9K/Defensin线性化后,运用氯化锂转化法将其转入巴斯德毕赤酵母GS115中,行G418加压筛选和PCR鉴定;所得阳性转化子转至摇瓶,28℃条件下0.5%甲醇诱导表达,连续诱导培养4 d后,上清液进行Tricine-SDS-PAGE检测表达效果,并采用琼脂糖孔穴扩散法检测表达产物对大肠杆菌E.coliK12D31的抑制作用。结果显示,家蝇抗菌肽Defensin在毕赤酵母中得到了成功表达,表达产物对大肠杆菌E.coliK12D31的生长有抑制作用。说明酵母系统适合抗菌肽的表达。     

Antimicrobial and Antibiofilm Activity of Mauriporin,a Multifunctional Scorpion Venom Peptide

Many pathogenic free living and biofilm forming bacterial organisms can cause serious infections to humans that could consequently have devastating effects on human health. A significant number of these microbial organisms are resistant to almost all known conventional antibiotics and the ability of some these strains to form sessile communities of biofilms increases the resistance ability of bacteria to antibiotic treatment. Global research is currently focused on finding novel therapies to counteract the threat of bacterial and biofilm infections rather than using conventional antibiotics. Mauriporin, a novel cationic α-helical peptide identified from the venom derived cDNA library of the scorpion Androctonus mauritanicus was reported to display selective cytotoxic and anti-proliferative activity against prostate cancer cell lines. In the present study, we investigated the antimicrobial and antibiofilm activities of Mauriporin. Our results show that Mauriporin displays potent antimicrobial activities against a range of Gram-positive and Gram-negative planktonic bacteria with MIC values in the range 5 µM to 10 µM. Mauriporin was also able to prevent Pseudomonas aeruginosa biofilm formation while showing weak hemolytic activity towards human erythrocytes. Studies on the mechanism of action of Mauriporin revealed that the peptide is probably inducing bacterial cell death through membrane permeabilization determined by the release of β-galactosidase enzyme from peptide treated Escherichia coli cells. Moreover, DNA binding studies found that Mauriporin can cause potent binding to intracellular DNA. All these results indicate that Mauriporin has a considerable potential for therapeutic application as a novel drug candidate for eradicating bacterial infections.     

Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells

The in vitro cytotoxicity of the antimicrobial peptide P40 was investigated. The food grade bacteriocin nisin was also analyzed for comparison. VERO cells were treated with different concentrations (0.02–2.5 μg ml⁻¹) of nisin and P40, and cell viability and plasma membrane integrity were checked by MTT, neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. In MTT and NRU assays the EC₅₀ to the purified peptide P40 were 0.30 and 0.51 μg ml⁻¹, while values found to nisin were 0.35 and 0.79 μg ml⁻¹, respectively. In the LDH assay, the EC₅₀ was 0.57 and 0.62 μg ml⁻¹ for P40 and nisin, respectively. The peptide P40 revealed higher hemolytical activity (19%) when compared to nisin (4.9%) at the highest concentration tested (2.5 μg ml⁻¹). Relatively few studies about the cytotoxicity of antimicrobial peptides are available. The determination of the cytotoxicity of antimicrobial peptides is an essential step to warrant their safe use.     

Antimicrobial Activity,Stability and Wound Healing Performances of Chitosan Nanoparticles Loaded Recombinant LL37 Antimicrobial Peptide

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides have illustrated potent abilities in the elimination of several pathogens resistant to conventional antimicrobial...     

Evaluation of Antimicrobial Activity of New Mastoparan Derivatives Using QSAR and Computational Mutagenesis

Antimicrobial peptides, also called body defense peptides, are used against a wide range of pathogens, such as negative- and positive-gram bacteria, mycobacteria, fungi, viruses, etc. Contrary to antibiotics, antimicrobial peptides do not develop resistance. Their wide antimicrobial spectrum situates them as important and attractive targets in research and pharmaceutical industry in order to obtain new structures using modern drug design techniques. We present here eleven QSAR models in which antimicrobial activity expressed as minimal inhibitory concentration values at Bacillus subtilis of 37 mastoparan analogs was correlated with different physicochemical parameters like: number of hydrophobic centers, molecular area and volume, internal dipole moment, refractivity, RPCG (relative positive charges) and number of donor and acceptor atoms generating by use of the computational software Sybyl. Significant R² (0.68–0.72) correlation coefficients and standard error of prediction SEE (0.199–0.230) were obtained, indicating that the established equations can be used. Thus, these linear models allowed us to create a library of 19 derivatives of mastoparan analogs obtained through computational mutagenesis. We propose this library of compounds as a source of possible derivatives with a more potent antimicrobial activity.     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

壳聚糖及其衍生物抗菌性质的研究进展

壳聚糖对多种细菌、真菌具有广谱抗菌的功能,因此它被广泛地应用于广泛地用于口腔疾病、皮肤炎症、伤口感染、胃肠道疾病等各种疾病的治疗。本文综述了壳聚糖及其衍生物对常见的口腔致病菌、皮肤癣菌、伤口感染菌以及胃肠道疾病的致病菌的抗菌作用和壳聚糖及其衍生物的抗菌机理。     

Solid-Phase Synthesis and Antibacterial Activity of an Artificial Cyclic Peptide Containing Two Disulfide Bridges

International Journal of Peptide Research and Therapeutics - Synthesis of artificial antibacterial peptides containing multiple disulfide bridges is of particular interest in bioorganic chemistry....     

Identification and Characterization of the Cecropin Antibacterial Protein Gene Locus in Drosophila virilis

Cecropin is a type of antibacterial peptide that is synthesized in response to infection and has been characterized in many insect species and one mammal. The Cecropin locus of Drosophila melanogaster also contains the gene Andropin, which has been identified only in this species and encodes a male-specific antibacterial peptide. As a first step in studying the molecular evolution of the cecropin and andropin genes among Drosophila species, we have isolated genomic clones that cover the Cecropin locus in Drosophila virilis. The cloned region totals approximately 25 kb, within which a 9-kb fragment contains four cecropin genes and one pseudogene. All four genes have a high level of sequence homology to D. melanogaster Cecropin, about 80% identity in the coding regions, and the intron positions are conserved. As in D. melanogaster and other insects, κB-related cis-regulatory elements are found upstream of these cecropin genes. An Andropin-related sequence was not identified in D. virilis; however, genome Southern hybridizations suggest that Andropin-related sequences are present in at least the melanogaster species subgroup. Analysis of 19 insect cecropin genes identifies a common ancestral Cecropin before the divergence of Diptera and Lepidoptera. In addition, D. melanogaster and D. virilis can be identified by monophyletic clades for Cecropin. In contrast, the Lepidopteran species show polyphyletic relationships for duplicated cecropin genes. Received: 12 August 1996 / Accepted: 18 October 1996     

Serial Expression and Activity Analysis of LNK-16: A Bovine Antimicrobial Peptide Analogue

Indolicidin is a broad-spectrum antimicrobial peptide (AMP) with great therapeutic potential; however, high manufacturing costs associated with industrial-scale chemical synthesis have limited its delivery. Therefore, the use of recombinant DNA technology to produce this peptide is urgently needed. In this study, a new methodology for the large-scale production of a novel bovine AMP was developed. LNK-16 is an analogue of indolicidin that contains a kallikrein protease site at its C-terminus. The amino acid sequence of LNK-16 was synthesized using Escherichia coli-preferred codons. Three copies of the target gene were assembled in series by overlapping PCR and cloned into pET-30a(+) for the expression of His-(LNK-16)₃ in E. coli BL21 (DE3) cells. The expressed fusion protein His-(LNK-16)₃ was purified by Ni²⁺-chelating chromatography and then cleaved by kallikrein to release LNK-16. The recombinant LNK-16 peptide showed antimicrobial activity similar to that of chemically synthesized LNK-16 and indolicidin. Together, these data indicate that the use of serial expression can improve the large-scale production of AMPs for clinical and research applications.     

一种兔肠源抗菌蛋白分离纯化及其抑菌活性

目的探讨兔肠道组织抗菌有效成分的组成及其性质。方法将新鲜兔小肠组织匀浆,经高温处理,乙酸浸提后,检测抗菌活性,再经Sephadex G100和Sephadex G75凝胶柱过滤层析,收集具有抗菌活性的蛋白组分,经SDS-PAGE电泳,考马斯亮蓝染色后,为一条蛋白带,其相对分子质量约为43×103。用琼脂糖弥散法和活菌计数实验检测纯化物对9株细菌的抑菌作用。结果分离得到一种纯的兔肠源抗菌蛋白,纯化的兔肠源抗菌蛋白对9株细菌的均有明显的抑菌作用,杀菌率介于78%与98%之间,显示了较强的抗菌活性。结论初步显示了兔肠源抗菌蛋白在细菌性疫病的防治方面的应用前景。