家蝇幼虫抗菌肽MDL-2的电泳制备及其抗菌作用机理

通过体壁损伤和感染大肠杆菌同时诱导家蝇Musca domestica幼虫产生免疫血淋巴,经沸水浴热变性,透析浓缩处理,然后经Tricine-SDS-PAGE得到诱导前后家蝇幼虫血淋巴中蛋白差异表达条带,将该条带电泳回收,复性,抗菌活性检测等步骤,分离纯化得到抗菌肽MDL-2,其分子中富含Pro,Gly和碱性氨基酸,分子量为11 kD,对革兰氏阴性菌Escherichia coli和革兰氏阳性菌Staphylococcus aureus均有较强抗性,因此电泳制备抗菌肽的方法为此类生物微量活性物质的分离纯化提供一种行之有效的途径。通过MDL-2对大肠杆菌和金黄色葡萄球菌通透性和透射电镜超微结构的图谱分析,MDL-2首先与细菌的外膜结合,然后抗菌肽形成柔性的两亲空间构象与细胞内膜作用,扰乱了膜脂分子的排列,改变了细胞膜的通透性,影响细胞膜的结构和功能,细胞膜上形成了许多孔道,同时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,最终引起细胞膜破碎,细胞完全解体,从而起到抑菌杀菌作用。     

一种源自人α-2-巨球蛋白的抗菌肽A2M3及其对金黄色葡萄球菌的抑菌机制

【目的】在人的鼻腔中鉴定出一种源自α-2-巨球蛋白的抗菌肽(命名为A2M3),并探究其对金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用和机制。【方法】结合生物信息学技术对人类鼻液的质谱结果进行分析,并筛选潜在抗菌肽;通过微量稀释法和平板涂布法分别分析A2M3对金黄色葡萄球菌最低抑菌浓度(minimum inhibitory concentration, MIC)和时间杀伤曲线(time-kill curve);采用透射电镜、碘化丙锭(propidium iodide, PI)摄取实验、流式细胞术和核酸蛋白质泄露实验分析A2M3对金黄色葡萄球菌膜完整性、膜通透性的影响;通过凝胶阻滞实验和荧光光谱实验探究A2M3对金黄色葡萄球菌基因组DNA的影响。【结果】利用生物信息学技术筛选出源自α-2-巨球蛋白的潜在抗菌肽A2M3,其对金黄色葡萄球菌的MIC为125.0 μg/mL,且能在3 h内完全杀灭细菌。A2M3通过增加细胞膜的通透性,促使核酸和蛋白质泄漏,继而穿过细胞膜嵌入DNA的碱基对,影响细菌的基因功能,从而导致菌体死亡。【结论】A2M3对金黄色葡萄球菌的抑菌机制涉及多靶点协同作用,能够改变细菌细胞膜的通透性,影响细菌的基因功能。这一发现揭示了从人体体液中筛选和分离抗菌功能肽的潜在应用价值。     

家蝇幼虫抗菌肽MDL-1的分离纯化及其对大肠杆菌超微结构的影响

通过体壁损伤和大肠杆菌同时诱导家蝇幼虫产生免疫血淋巴,经沸水浴热变性,透析浓缩处理,然后经Tricine-SDS-PAGE得到诱导前后家蝇幼虫血淋巴中蛋白差异表达条带,将该条带电泳回收、复性、抗菌活性检测等步骤,分离纯化得到抗菌肽MDL-1, 其分子中富含Gly和碱性氨基酸,分子量为6 200 D,对革兰氏阴性菌Escherichia coli有较强抗性。通过MDL-1对大肠杆菌通透性分析和透射电镜超微结构观察表明,MDL-1首先可能与细菌的外膜结合,然后与细胞内膜作用,扰乱膜脂分子的排列,改变细胞膜的通透性,从而影响细胞膜的结构和功能,使细胞膜形成的许多孔道,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,最终引起细胞膜破碎,细胞完全解体,从而起到抑菌杀菌作用。     

纳米颗粒抗菌机理的研究进展

随着具有抗菌效应的纳米颗粒被大量报道,纳米颗粒的抑菌杀菌机理也成为重要的研究领域并取得一定进展,本文综述了常见纳米颗粒作用机理的研究进展。当前大多数实验表明,纳米颗粒引起细胞膜的破坏是其抗菌抑菌效应的主要原因,结合已有研究,作者提出,纳米颗粒抑菌杀菌分为四个阶段:同细胞的接触、与细胞膜的相互作用及对膜的破坏、胞内杀菌和细菌死亡。文中重点分析探讨了纳米颗粒同细菌细胞膜作用过程中一些待解答的基础性问题。最后通过比较发现,纳米颗粒同抗生素作用方式相异,而与抗菌肽的作用模式相近,细菌对纳米颗粒较难产生耐药性,这对当前治疗耐药菌株的感染有良好的前景。     

载银纳米抗菌复合骨填充材料体外抗菌性能研究

目的探讨新型载银纳米抗菌复合骨填充材料(TiO2-Ag-nHA/PA66)的体外抗菌性能。方法采用抑菌环试验及菌落总数测定法检测不同纳米抗菌复合骨填充材料(A1、A2、A3)对金黄色葡萄球菌及大肠埃希菌的体外抗菌效果;扫描电镜观察其对细菌的抗粘附作用。结果抑菌环试验显示,不同载银纳米抗菌复合骨填充材料对金黄色葡萄球菌和大肠埃希菌均形成明显的抑菌环,以作用24 h抑菌环直径最大,并随作用时间延长,抑菌环直径逐渐缩小。其中银含量为0.64%(质量比)的材料A3的抗菌作用最明显,持续时间最长,其对金黄色葡萄球菌和大肠埃希菌的抑菌作用持续时间分别达到33 d和24 d;菌落总数测定法显示细菌与材料A3接触24 h后,对金黄色葡萄球菌和大肠埃希菌的抗菌率分别为94.18%和85.96%;扫描电镜发现载银材料能够明显减少细菌在材料表面的粘附。结论载银纳米抗菌复合骨填充材料体外对金黄色葡萄球菌及大肠埃希菌有明显抗菌作用,为其应用于慢性骨髓炎术后骨缺损修复提供理论依据。     

条斑紫菜蛋白酶解产物中抗菌肽的纯化及特征

以酶解产物对金黄色葡萄球菌的抑菌能力为指标,筛选出木瓜蛋白酶并优化确定酶解条件,酶解液中分子量在1000 Da以下的小分子肽类占96.68%;采用DEAE-52纤维素阴离子交换层析、Sephadex G-25凝胶层析得到纯化的紫菜蛋白抗菌肽(LPAP);液质联用分析该纯化抗菌肽中主要的多肽序列为FFDD(Phe-Phe-AspAsp);该LPAP对G+菌和G-菌都有抑制作用,具有相对广谱性,对金黄色葡萄球菌的最小抑菌浓度(Minimum inhibitory concentration,MIC)为0.25 mg/m L,抗菌肽作用金黄色葡萄球菌的电镜照片表明其抑菌机制可能是对细胞膜的破坏作用。     

家蝇幼虫抗菌肽MDL-2对细菌细胞渗透性及代谢功能影响

研究了家蝇幼虫抗菌肽MDL-2与细菌相互作用时,抗菌肤MDL-2对细菌细胞壁的溶解作用、细胞膜渗透性和代谢的影响.抗菌肽MDL-2在抗菌过程中首先与细菌的细胞壁相互作用,使其破裂,抗菌肽对革兰氏阴性细菌大肠杆菌细胞壁的作用有浓度依赖性,而对革兰氏阳性细菌金黄色葡萄球菌MDL-2在较低的浓度时即可发生细胞壁破坏作用;抗菌...     

少棘蜈蚣抗菌肽Scolopin 2-NH2的抗菌作用机制研究

旨在探究抗菌肽Scolopin-2(S-2)及其酰胺化修饰产物Scolopin-2-NH2(S-2-N)的二级结构、抗菌活性及其理化性质,并从细胞水平和分子水平揭示其抗菌作用机制。按最小抑菌浓度测定法检测抗菌肽S-2/S-2-N对4种菌株的抗菌活性;用不同条件处理S-2/S-2-N,测定其理化性质;用圆二色谱仪检测抗菌肽S-2/S-2-N的二级结构;以S-2-N/S-2处理大肠杆菌,用流式细胞仪检测二者穿透细胞膜的效率及菌体细胞膜完整性;通过流式细胞术和RT-PCR技术分析S-2-N/S-2对细菌细胞周期及细胞内DNA复制和修复相关基因的影响。除了具有和母体肽S-2相似的耐热性、耐酸碱性及抵抗离子强度和消化酶的能力,S-2-N还具有更强的抗菌活性;流式细胞仪检测结果显示,短时间内(30 min)二者均能引起一定程度的细胞膜损伤,但抗菌肽S-2-N穿透细胞膜的效率更高;流式细胞术及RT-PCR技术分析结果显示,S-2-N/S-2抑制了大肠杆菌细胞周期的进行,下调了基因dnaA、dnaB、dnaG和SSB的表达,同时促进了RecA和RecN的表达。抗菌肽S-2经酰胺化修饰后,具有比母体肽更强的抗菌活性。二者的抗菌作用机制为:破坏细菌细胞膜,结合细菌DNA和RNA、影响DNA的二级结构,阻滞细菌细胞周期的进行、影响细菌细胞内与DNA复制和修复相关基因的表达。     

枯草芽孢杆菌HS-A38高产抗菌肽突变株的筛选及其抑菌机理的研究

本研究通过采用多种诱变方法以提高枯草芽孢杆菌HS-A38产抗菌肽能力,并研究诱变株产抗菌肽活性物质对副溶血弧菌的抑制作用机理。原始菌株经过多种复合诱变后,最终获得一株抗菌活性明显提高的诱变菌株(mut HS-301)。进一步研究采用双倍稀释法确定最低抑菌浓度,并通过扫描电镜观察、胞膜通透性和胞膜完整性的测定,初步阐述该抗菌肽的抑菌机理。结果表明:诱变菌株mut HS-301对副溶血弧菌的抑菌活性较原始菌株提高了20%;抗菌肽成品对副溶血弧菌的最低抑菌浓度为0.625 mg/m L;抗菌肽破坏了菌体细胞的形态,出现形变;随着抗菌肽作用时间的增加,胞外紫外吸收物质和胞外蛋白含量明显增加,说明抗菌肽影响了副溶血弧菌细胞膜的通透性,其胞膜的完整性遭到破坏,影响其代谢活性,从而抑制副溶血弧菌的生长。该研究表明,微生物产抗菌肽主要作用于细菌细胞膜,该产品适宜添加到水产动物养殖饲料中,对提高水产动物免疫力和预防疾病起重要作用。     

抗菌肽抗菌机制及其应用研究进展

抗菌肽是一类小分子肽,具有广谱的抗菌活性。以往对抗菌肽抗菌机制的研究主要集中在细菌细胞膜的作用上,包含"桶板"模型、"毯式"模型,"环形孔"模型和"凝聚"模型。近年来相继发现某些抗菌肽可以作用于细菌细胞内部,与核酸物质结合,阻断DNA复制、RNA合成;影响蛋白质合成;抑制隔膜、细胞壁合成,阻碍细胞分裂;抑制胞内酶的活性。本文从胞内机制和胞外机制两个角度对抗菌肽的抗菌机制进行综述,以期阐明各类抗菌肽的作用机制,为进一步研究菌株耐药性、杀菌效果及其杀菌机制提供科学根据。     

Antimicrobial action of novel chitin derivative

Aminoethyl-chitin (AEC) was synthesized in an attempt to both increase solubility of chitin in water and biological activity. AEC was obtained by grafting 2-chloroethylamino hydrochloride onto chitin at C-6 position. The structure of AEC was elucidated FT-IR and (1)H NMR spectroscopy, and its antimicrobial activity was investigated using three Gram-positive and Gram-negative bacteria. The integrity of the cell membranes of the representatives E. coli and S. aureus was investigated by determining the release of intracellular components of cells. When treated with AEC, release of 260 nm absorbing materials quickly increased both E. coli and S. aureus, but absorbance value was different due to the difference in cell structures. For detailed study, outer membrane (OM) and inner membrane (IM) permeabilization assay were performed using the fluorescent probe 1-N-phenylnaphthylamine (NPN) and the release of cytoplasmic beta-galactosidase activity. The results showed that AEC rapidly increased NPN uptake and the release of cytoplasmic beta-galactosidase via increasing the permeability of OM and IM. In addition, cytotoxic effect of AEC was assessed using human lung fibroblast (MRC-5) cell line, and AEC showed less toxic against MRC-5.     

Antimicrobial activity of Brazilian copaiba oils obtained from different species of the Copaifera genus

The antimicrobial activity of copaiba oils was tested against Gram-positive and Gram-negative bacteria, yeast, and dermatophytes. Oils obtained from Copaifera martii, Copaifera officinalis, and Copaifera reticulata (collected in the state of Acre) were active against Gram-positive species (Staphylococcus aureus, methicillin-resistant S. aureus, Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus faecalis) with minimum inhibitory concentrations ranging from 31.3-62.5 microg/ml. The oils showed bactericidal activity, decreasing the viability of these Gram-positive bacteria within 3 h. Moderate activity was observed against dermatophyte fungi (Trichophyton rubrum and Microsporum canis). The oils showed no activity against Gram-negative bacteria and yeast. Scannning electron microscopy of S. aureus treated with resin oil from C. martii revealed lysis of the bacteria, causing cellular agglomerates. Transmission electron microscopy revealed disruption and damage to the cell wall, resulting in the release of cytoplasmic compounds, alterations in morphology, and a decrease in cell volume, indicating that copaiba oil may affect the cell wall.     

Poly(ethylene imine)s as Antimicrobial Agents with Selective Activity

We report the structure-activity relationship in the antimicrobial activity of linear and branched poly(ethylene imine)s (L- and B-PEIs) with a range of molecular weights (MWs) (500-12?000). Both L- and B-PEIs displayed enhanced activity against Staphylococcus aureus over Escherichia coli. Both B- and L-PEIs did not cause any significant permeabilization of E. coli cytoplasmic membrane. L-PEIs induced depolarization of S. aureus membrane although B-PEIs did not. The low MW B-PEIs caused little or no hemolysis while L-PEIs are hemolytic. The low MW B-PEIs are less cytotoxic to human HEp-2 cells than other PEIs. However, they induced significant cell viability reduction after 24 h incubation. The results presented here highlight the interplay between polymer size and structure on activity.     

Membrane Disruption by Antimicrobial Fatty Acids Releases Low-Molecular-Weight Proteins from Staphylococcus aureus

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward Gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1Δ9 was used for phospholipid synthesis, whereas toxic 16:1Δ9 was not and required elongation to 18:1Δ11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1Δ9 was as toxic as 16:1Δ9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus.     

Antimicrobial activity and synergism of some substituted flavonoids

Natural and synthetic substituted chalcones, flavones and flavanones were tested for antibacterial activity. In order to determine synergism, new combinations of substituted flavonoids against Staphylococcus aureus, Escherichia coli and Enterobacter aerogenes were assayed. The results allow us to establish relationships between antimicrobial effect of the compounds and membrane structures of these microorganisms. When flavonoid combinations were employed a stronger effect was found against E. coli than against S. aureus. This fact is due to the existence of porins in the outer membrane of G(-)-bacteria. The compound that acts as enhancer acts by blocking the charges of amino acids in the porins and thus facilitates the passage of the other compound by diffusion into the bacterial cell.     

鱼精蛋白抗菌机制的研究

鱼精蛋白是一种存在于各类动物精巢组织中的多聚阳离子肽,其抗菌性很早就被所知,然而它的抗菌机理却一直未能得到很清楚的了解。现存在的机理有2种:一种认为鱼精蛋白与细菌细胞壁结合,通过破坏细胞壁的形成来达到抑菌效果;另一种认为鱼精蛋白破坏了细胞能量的转换、营养物质的吸收功能,细胞质膜是鱼精蛋白攻击的对象。事实上,作者认为,鱼精蛋白的抗菌效果可能是通过以上2种方式共同作用的结果,因而它的抗菌机理也可能是这两种机理的叠加,这还需进一步的研究证明。     

Antimicrobial activity of a chimeric enzybiotic towards Staphylococcus aureus

Phage lytic enzymes (enzybiotics) have gained attention as prospective tools to eradicate Gram-positive pathogens resistant to antibiotics. Attempts to purify the P16 endolysin of Staphylococcus aureus phage P68 were unsuccessful owing to the poor solubility of the protein. To overcome this limitation, we constructed a chimeric endolysin (P16-17) comprised of the inferred N-terminal d-alanyl-glycyl endopeptidase domain and the C-terminal cell wall targeting domain of the S. aureus phage P16 endolysin and the P17 minor coat protein, respectively. The domain swapping approach and the applied purification procedure resulted in soluble P16-17 protein, which exhibited antimicrobial activity towards S. aureus. In addition, P16-17 augmented the antimicrobial efficacy of the antibiotic gentamicin. This synergistic effect could be useful to reduce the effective dose of aminoglycoside antibiotics.     

Mechanisms of action of acriflavine: electron microscopic study of cell wall changes induced in Staphylococcus aureus by acriflavine

The antimicrobial action of acriflavine, a quaternary ammonium compound, on Staphylococcus aureus was studied by electron microscopic observation. The bactericidal activity of acriflavine was dose-dependent over the 4 hr of exposure time. Scanning electron micrographs showed a wavy wrinkled cell surface following treatment with acriflavine. Transmission electron micrographs showed thickened cell walls following treatment with acriflavine. Acriflavine-induced cell wall thickness seemed to affect both the peripheral and cross walls, but was reversible after treatment removal. These findings indicate that cell wall thickness is a characteristic phenotype of S. aureus exposed to acriflavine. It is therefore believed that cell wall thickness plays an important role in the mechanism of action of acriflavine.     

Antimicrobial activity of tertiary amine covalently bonded to a polystyrene fiber.

Tertiary amine was covalently bonded to a polystyrene fiber and examined for antibacterial activity. The tertiary amine covalently bonded to a polystyrene fiber (TAF) showed a high antimicrobial activity against Escherichia coli. TAF exhibited a stronger antibacterial activity against gram-negative bacteria (E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens) than against gram-positive bacteria (Staphylococcus aureus and Streptococcus faecalis) or Candida albicans. This activity against E. coli was accentuated by 0.1% deoxycholate or 10 mg of actinomycin D per ml, to which E. coli is normally not susceptible. This implies that TAF causes an increase of the bacterial outer membrane permeability. On the other hand, the antimicrobial activity was inhibited by adding Mg2+ or by lowering the pH. This suggest an electrostatic interaction between the bacterial cell wall and TAF. Scanning electron microscopy showed that E. coli cells were initially attached to TAF, with many projections on the cell surface, but then were apparently lysed after contact for 4 h. Taken together, these results imply that bacteria initially interact with TAF by an electrostatic force between the anionic bacterial outer membrane and the cationic tertiary amine residues of TAF and that longer contact with TAF damages the bacterial outer membrane structure and increases its permeability.