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.     

Rapid Cytotoxicity of Antimicrobial Peptide Tempoprin-1CEa in Breast Cancer Cells through Membrane Destruction and Intracellular Calcium Mechanism

Temporin-1CEa is an antimicrobial peptide isolated from the skin secretions of the Chinese brown frog (Rana chensinensis). We have previously reported the rapid and broad-spectrum anticancer activity of temporin-1CEa in vitro. However, the detailed mechanisms for temporin-1CEa-induced cancer cell death are still weakly understood. In the present study, the mechanisms of temporin-1CEa-induced rapid cytotoxicity on two human breast cancer cell lines, MDA-MB-231 and MCF-7, were investigated. The MTT assay and the LDH leakage assay indicated that one-hour of incubation with temporin-1CEa led to cytotoxicity in a dose-dependent manner. The morphological observation using electronic microscopes suggested that one-hour exposure of temporin-1CEa resulted in profound morphological changes in both MDA-MB-231 and MCF-7 cells. The membrane-disrupting property of temporin-1CEa was further characterized by induction of cell-surface exposure of phosphatidylserine, elevation of plasma membrane permeability and rapid depolarization of transmembrane potential. Moreover, temporin-1CEa evoked intracellular calcium ion and reactive oxygen species (ROS) elevations as well as collapse of mitochondrial membrane potential (Δφm). In summary, the present study indicates that temporin-1CEa triggers rapid cell death in breast cancer cells. This rapid cytotoxic activity might be mediated by both membrane destruction and intracellular calcium mechanism.     

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...     

Strong Synergy between a Eukaryotic Antimicrobial Peptide and Bacteriocins from Lactic Acid Bacteria

The antimicrobial effect obtained upon combining the prokaryotic antimicrobial peptides (AMPs; more commonly referred to as bacteriocins) pediocin PA-1, sakacin P, and curvacin A (all produced by lactic acid bacteria [LAB]) with the eukaryotic AMP pleurocidin (from fish) has been investigated. The three LAB AMPs alone were active against gram-positive Listeria ivanovii bacteria at nanomolar concentrations, whereas they were inactive against gram-negative Escherichia coli bacteria. Pleurocidin alone was active against both of these types of bacteria at micromolar concentrations. Little if any synergy between the LAB AMPs and pleurocidin against the gram-positive L. ivanovii strain was obtained. In contrast, the LAB AMPs and pleurocidin acted highly synergistically against the gram-negative E. coli strain. Nanomolar concentrations of LAB AMPs increased the growth inhibitory potency of pleurocidin by about fourfold. When micromolar concentrations of LAB AMPs were combined with 2 μg of pleurocidin/ml, 100% growth inhibition was attained, whereas pleurocidin alone at a concentration of 2 μg/ml gave no growth inhibition. Most noteworthy, when high concentrations (128 μg/ml) of pleurocidin in the absence of LAB AMPs were used over a long period of incubation (1 week), some growth of E. coli was observed, whereas 16 μg of pleurocidin/ml completely abolished growth in the presence of 64 to 128 ng of LAB AMPs/ml over the same period of time. The results clearly demonstrate that combining eukaryotic and prokaryotic AMPs can greatly increase the specific activity and broaden the target-cell range of these peptides.     

Bombesin Analogue-Mediated Delivery Preferentially Enhances the Cytotoxicity of a Mitochondria-Disrupting Peptide in Tumor Cells

Tumor-homing peptides that recognize specific markers on tumor cells have shown potential as drug carriers for targeted cancer therapy. Bombesin receptors are frequently overexpressed or ectopically expressed in a wide range of human tumors. Bombesin and its analogues have been widely used as drug carriers for tumor imaging and tumor therapy. However, the cargos used in previous studies, including radioactive and chemotherapeutic agents, are usually small molecules. Mitochondrial-disrupting peptides depolarize the mitochondria and trigger apoptosis after entering tumor cells. We are interested in whether the bombesin analogue, Bn(6–14), which contains a bombesin receptor-binding motif, can specifically deliver the mitochondria-disrupting peptide, B28, to tumor cells. To this end, we created a chimeric peptide, B28Bn(6–14), by conjugating B28 to Bn(6–14) at its N-terminus. The cytotoxicity of B28Bn(6–14) in tumor cells was much stronger than unconjugated B28. The IC₅₀ values of B28Bn(6–14) in tumor cells (1.7–3.5 µM) were approximately 10 times lower than B28. However, conjugation of B28 to Bn(2–7), which lacks the bombesin receptor-binding motif, did not increase its cytotoxicity. In addition, the IC₅₀ values of B28Bn(6–14) in tumor cells (1.7–3.5 µM) was 3–10 times lower than in normal cells (10.8–16.8 µM). We found that selective binding of B28Bn(6–14) to tumor cells is Bn(6–14)-dependent. Upon entering the tumor cell, B28Bn(6–14) accumulated in the mitochondria and triggered caspase-dependent apoptosis. Intratumoral and intraperitoneal administration of B28Bn(6–14) substantially suppressed the growth of DU145 tumor xenografts in mice. These results demonstrate that Bn(6–14) is able to deliver the mitochondria-disrupting peptide to tumor cells, and B28Bn(6–14) should be further developed as novel anti-cancer agent.     

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.     

抗菌肽OH-CATH对大肠杆菌Escherichia coli的作用(英文）

OH-CATH是眼镜王蛇中新发现的cathelicidin家族抗菌肽.它在1%NaCI存在的条件下对多种细菌都有较强的抗菌活性,同时,在高浓度下对人红细胞无溶血活性.OH-CATH足开发新型抗菌药物的优良模板.蜊明OH-CATH的作用机理及其对微生物的选择性,对研发以OH-CATH为先导结构的药物研发有十分重要的意义.本文利用扫描电镜以及透射电镜对OH-CATH与革兰氏阴性菌一大肠杆菌ATCC 25922相互作用的效应研究.结果揭示:OH-CATH对大肠杆菌的作用涉及到3个步骤.首先,OH-CATH借助其带正电的氨基酸残基附着到细菌带负电荷的细胞壁:然后,附着的OH-CATH在达剑一定浓度后发生聚集,以孔道彤成的方式破坏细菌的膜结构;最终,由十细菌膜的损坏,膜的渗透性被破坏,胞内内含物释放造成细菌死亡.     

抗菌肽OH-CATH对大肠杆菌Escherichia coli的作用

OH-CATH是眼镜王蛇中新发现的cathelicidin家族抗菌肽.它在1%NaCI存在的条件下对多种细菌都有较强的抗菌活性,同时,在高浓度下对人红细胞无溶血活性.OH-CATH足开发新型抗菌药物的优良模板.蜊明OH-CATH的作用机理及其对微生物的选择性,对研发以OH-CATH为先导结构的药物研发有十分重要的意义.本文利用扫描电镜以及透射电镜对OH-CATH与革兰氏阴性菌一大肠杆菌ATCC 25922相互作用的效应研究.结果揭示:OH-CATH对大肠杆菌的作用涉及到3个步骤.首先,OH-CATH借助其带正电的氨基酸残基附着到细菌带负电荷的细胞壁:然后,附着的OH-CATH在达剑一定浓度后发生聚集,以孔道彤成的方式破坏细菌的膜结构;最终,由十细菌膜的损坏,膜的渗透性被破坏,胞内内含物释放造成细菌死亡.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Pro-Inflammatory Cytokine Responses of A549 Epithelial Cells to Antimicrobial Peptide Brevinin-2R

Brevinin-2R is an antimicrobial peptide which has been isolated from the skin of the frog Rana ridibunda. The purpose of the present study was to examine the cellular cytotoxicity and inflammatory effects of brevinin-2R (B2R) on human lung epithelial adenocarcinoma cell line A549. The effects of different concentrations (5, 10, and 20 μg/ml) of B2R on the expression levels of pro-inflammatory cytokines such as IL-1β, and IL-8 in A549 cells were evaluated by semi-quantitative RT-PCR and real-time PCR assays in a dose- and time-dependent manner. Based on the results of MTT assay, B2R showed a moderate cytotoxicity effect in a dose-dependent manner up to 20 % suppression of the cell growth. Moreover, gene expression results demonstrated that B2R up-regulates the IL-1β and IL-8 expression levels in A549 cells in a dose- and time-dependent manner. Our results suggested that brevinin-2R antimicrobial peptide has potentially a regulatory effect on triggering the inflammatory processes.     

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.     

Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.     

真核表达MERS-CoV刺突蛋白亚单位的信号肽序列优化研究

中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的刺突蛋白(Spike,S)亚单位1(S1)是引起宿主免疫反应和产生中和抗体的主要靶抗原,也是疫苗研发和病原检测的重要靶标,选用适宜的真核表达系统高效表达S1蛋白是进行相关研究的基础。为确定MERS-CoV S1在哺乳动物细胞中高效分泌性表达的信号肽序列,构建了含高斯荧光素酶(Gaussia luciferase,GLuc)、人组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)及小鼠免疫球蛋白G的2a亚型(Mouse immunoglobular G subtype 2a,MIgG2a)7个信号肽(原始序列和改造序列)序列的MERS-CoV S1表达质粒,瞬时转染细胞后,通过Western Blot检测并比较细胞培养上清和裂解液中S1的表达水平及分泌表达效率(条带密度灰度扫描比),并对哺乳动物细胞表达的S1蛋白的纯度与抗原特性进行了分析。结果表明7种信号肽在293T、BHK21和ExpiCHO-S^TM三种细胞系统中介导MERS-CoV S1的高效分泌表达的效率各有不同,其中tPA-1信号肽介导S1抗原在ExpiCHO-S^TM中具有较高的分泌表达效率与产量,纯化的S1蛋白保持了较好的抗原性。本研究为进一步研发基于MERS-CoV S1的亚单位疫苗及免疫学检测试剂奠定了基础。     

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。     

牛气管黏膜抗菌肽成熟肽基因的克隆及序列分析

目的:获得牛气管黏膜抗菌肽(bTAP)成熟肽的基因序列,为后续的研究工作奠定基础。方法:从新屠宰的黄牛气管黏膜中提取总RNA,反转录获得cDNA,以此cDNA为模板进行PCR扩增目的片段,并将其克隆至pMD18-T载体中,经鉴定随机挑选1个阳性重组子进行测序,将测序结果与已报道的序列进行比较,并做NCBIBlast比对。结果:PCR扩增出bTAP成熟肽基因,核苷酸序列测定验证了其正确性;NCBIBlast比对表明,与bTAP成熟肽基因同源性较高的分别是牛β-防御素11、牛β-防御素12、牛β-防御素402、牛β-防御素403、绵羊β-防御素1、绵羊β-防御素2、山羊β-防御素1及山羊β-防御素2,核苷酸序列同源性分别为78.07%、78.95%、80.70%、83.33%、83.33%、80.70%、81.58%和81.58%,氨基酸序列同源性分别为68.42%、65.79%、68.42%、76.32%、71.05%、63.16%、63.16%和68.42%。结论:成功克隆了bTAP成熟肽的基因序列,NCBIBlas比对表明bTAP与防御素可能来自一个共同的祖系基因。     

Alanine Scanning Studies of the Antimicrobial Peptide Aurein 1.2

Antimicrobial peptides (AMPs) are compounds widely distributed in nature that display activity against a broad spectrum of pathogens. Amphibian skin, as an organ rich in pharmacologically active peptides, appears to be an interesting source of novel AMPs. Aurein 1.2 (GLFDIIKKIAESF-NH₂) is a short 13-residue antimicrobial peptide primarily isolated from the skin secretions of Australian bell frogs. In this study, the alanine scan of aurein 1.2 was performed to investigate the effect of each amino acid residue on its biological and physico-chemical properties. The biological studies included determination of minimum inhibitory concentration, activity against biofilm, and inhibitory effect on its formation. Moreover, the hemolytic activity as well as serum stability was determined. The hydrophobicity of peptides and their self-assembly were investigated using reversed-phase chromatography. In addition, their helicity was calculated from circular dichroism spectra. The results not only provided information on structure-activity relationship of aurein 1.2 but also gave insights into design of novel analogs of AMPs in the future.

     

APP5肽模拟物P165对体外培养大鼠海马神经干细胞增殖和分化的影响

目的研究一种小分子多肽─APP5肽的模拟物P165对体外培养的大鼠胚胎海马神经干细胞（neuralstem cells,NSCs）增殖和分化的影响,以期能找到一种可代替神经营养因子的小分子物质,能够促进NSCs的增殖或分化,为将来的临床应用提供理论依据。方法（1）原代培养SD大鼠胚胎脑海马NSCs;（2）利用5-溴脱氧尿嘧啶核苷（BrdU）和神经元、星型胶质细胞、少突胶质细胞的特异性标记物微管相关蛋白2（MAP2）、胶质纤维酸性蛋白（GFAP）、2,3-环核苷酸-3磷酸二酯酶（CNPase）对培养的NSCs进行鉴定;（3）将培养的NSCs分为对照组、血清组、APP5肽反序列组和P165组,观察各组细胞形态的变化;（4）将培养的NSCs分为对照组、APP5肽反序列组和P165组,利用细胞计数,测定干细胞克隆形成率、干细胞克隆形成大小的方法分析P165对海马NSCs增殖的影响。结果（1）海马神经干细胞呈神经球聚集生长,BrdU染色阳性;加入血清后神经球周围有细胞呈放射状向四周生长,并带有突起。染色呈MAP2、GFAP或CNPase阳性;（2）海马NSCs加入P165及其反序列后细胞形态上与对照组相比没有明显改变;（3）与对照组相比,加P165后海马NSCs数量明显增加,克隆形成率和克隆形成的直径均有明显的增加,并有统计学差异。结论P165能够促进海马NSCs的增殖,但并不促进其分化。     

Identification of OmpT as the Protease That Hydrolyzes the Antimicrobial Peptide Protamine before It Enters Growing Cells of Escherichia coli

The influence of extracytoplasmic proteases on the resistance of Escherichia coli to the antimicrobial peptide protamine was investigated by testing strains with deletions in the protease genes degP, ptr, and ompT. Only ΔompT strains were hypersusceptible to protamine. This effect was abolished by plasmids carrying ompT. Both at low and at high Mg²⁺ concentrations, ompT⁺ strains cleared protamine from the medium within a few minutes. By contrast, at high Mg²⁺ concentrations, protamine remained present for at least 1 h in the medium of an ompT strain. These data indicate that OmpT is the protease that degrades protamine and that it exerts this function at the external face of the outer membrane.