家蝇幼虫抗菌肽的抗菌谱及其与抗生素的协同作用研究

研究3种家蝇幼虫抗菌肽的抗菌谱以及每种抗菌肽的最小抑菌浓度(MIC),初步探讨3种抗菌肽分别与青霉素、链霉素相结合后对抗菌活性的影响,并采用分级抑制浓度指数(Fractionalinhibitoryconcentrationindex,FIC)来定量检测抗菌肽与抗生素之间的抗菌作用关系。结果表明3种抗菌肽的抗菌谱不同,不同的抗菌肽对不同病原菌的抗菌活性不同。3种抗菌肽与链霉素、青霉素之间的抗菌协同关系因细菌种类不同。抗菌肽与抗生素之间并不是都存在协同关系,有些不相关,甚至表现为对抗关系,表明抗菌肽、抗生素与细菌三者的相互作用关系非常复杂。     

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。     

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。     

柞蚕蛹抗菌肽D对大肠杆菌K_(12)D_(31)的作用

本文报道了不同浓度的柞蚕蛹抗菌肽D,对大肠杆菌K_(12)D_(31)的杀灭作用动力学。在LEG培养液中,抗菌从D的浓度在5微克/毫升时显效。浓度在10微克/毫升以上时,其杀菌速度大于细菌的增殖速度。固定抗菌肽浓度为10微克/毫升,细菌浓度在3×10~7个细菌/毫升,培养在磷酸钾盐缓冲液中,4小时后能全部杀灭。同样浓度细菌在LEG培养液中,4小时后细菌数下降到约为10~2个细菌/毫升,但不能全部杀灭。同时还提供了柞蚕蛹抗菌肽D和B对大肠杆菌K_(12)D_(31)作用不同时间的电镜照片。     

家蝇幼虫抗菌肽MDL-3分子结构的荧光特性

研究了家蝇幼虫抗菌肽MDL-3的荧光光谱和淬灭剂对内源性荧光的影响.家蝇幼虫抗菌肽MDL-3在280nm波长的激发光时,荧光光谱为Tyr残基和Trp残基共同提供,KI不能淬灭抗菌肽MDL-3的Trp残基的荧光,而Acr只能淬灭71%(f=0.71)的抗菌肽MDL-3中的Trp残基的荧光,说明Trp残基不是位于抗菌肽分子的表面,而是位于分子的内部.     

抗菌肽对细菌杀伤作用的分子机制

抗菌肽是一类新型的抗菌物质,从最低等的生物病毒、细菌到高等的动植物都有广泛分布. 以往的研究主要集中于抗菌肽对细菌细胞膜的作用机制,已经构建了三种作用模式. 但近几年的研究表明,很多抗菌肽都能有效地穿过细菌的细胞膜,直接与胞内分子相互作用,并不引起膜的破裂. 抗菌肽根据其结构特点有着多种杀菌穿膜的机制,其后分别与胞内的靶分子如核酸,蛋白质,信号转导通路等互相作用,最终实现对细菌的杀伤作用.     

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。     

某些抗菌肽的研究进展

抗菌肽是各种生物防御系统的一个组成部分,是由于微生物等因素的侵染而产生的免疫应答反应产物,具有分子量低,热稳定,强碱性和广谱抗菌等特点,抗菌肽的正电荷与细菌细胞的磷脂头负电荷之间的相互作用是重要,肽分子氮端的两性螺旋是裂解细菌的主要部分,碳末端酰胺化与抗菌肽的广谱抗菌有关,抗菌肽具有独特的抗菌机理,能在细菌质膜上形成离子通道,破坏膜势,引起胞内物质泄漏,从而杀灭细菌。     

新疆家蚕抗菌肽抗菌作用的超微结构观察及抗菌机理初探

为探讨基因工程表达的新疆家蚕(Bombyx mori)抗菌肽(cecropin-XJ)的抗菌机制,通过紫外分光光度法研究抗菌肽的抑菌动力学,并采用透射电镜观察抗菌肽作用于金黄色葡萄球菌(Staphylococcus aureus)后的超微结构,对抗菌肽抗菌机理进行初步探讨。结果表明,抗菌肽抑菌作用比较明显,抗菌肽的活性与作用时间有关。抗菌肽可能是通过"桶-板"模式渗透细胞膜,从而影响细胞膜的结构和功能,使细胞膜形成许多孔道,增强了金黄色葡萄球菌细胞的通透性,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,从而起到抑菌、杀菌作用。抗菌肽使金黄色葡萄球菌细胞内容物大量渗漏而死亡,死亡细胞的细胞壁保持完整,表明细胞膜是抗菌肽作用的主要靶位点。     

细菌细胞壁生长调控机制研究进展

在细菌生长过程中,细胞壁起到维持细胞形状和完整性,抵抗内部膨胀压的作用。细胞壁的合成、分裂、再生、循环再利用等与细菌自身生长繁殖和应对环境压力息息相关。目前,细胞壁生长机理,细菌如何调控细胞壁生长及如何与其他细胞过程相协调的机制尚未研究清楚。细胞壁调控机制的解析对了解细菌细胞壁功能、确定药物的作用方式和发展新一代的治疗方法至关重要。对细菌调控细胞壁生长机制的国外研究进展进行了概述,重点阐述了支架蛋白、转录因子、非编码小RNA及蛋白相互作用调控细胞壁的合成、细胞分裂、压力响应的机制,总结了细胞壁调控机制在抗菌药物研发中的应用,并对未来的研究方向进行了展望。     

Antibacterial activity and mechanism of a scorpion venom peptide derivative in vitro and in vivo

BmKn2 is an antimicrobial peptide (AMP) characterized from the venom of scorpion Mesobuthus martensii Karsch by our group. In this study, Kn2-7 was derived from BmKn2 to improve the antibacterial activity and decrease hemolytic activity. Kn2-7 showed increased inhibitory activity against both gram-positive bacteria and gram-negative bacteria. Moreover, Kn2-7 exhibited higher antibacterial activity against clinical antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). In addition, the topical use of Kn2-7 effectively protected the skin of mice from infection in an S. aureus mouse skin infection model. Kn2-7 exerted its antibacterial activity via a bactericidal mechanism. Kn2-7 killed S. aureus and E. coli rapidly by binding to the lipoteichoic acid (LTA) in the S. aureus cell wall and the lipopolysaccharides (LPS) in the E. coli cell wall, respectively. Finally, the hemolytic activity of Kn2-7 was significantly decreased, compared to the wild-type peptide BmKn2. Taken together, the Kn2-7 peptide can be developed as a topical therapeutic agent for treating bacterial infections.     

羟基酪醇抑菌活性及抑菌稳定性研究

本研究探究了羟基酪醇对大肠杆菌、金黄色葡萄球菌、铜绿假单胞杆菌和枯草芽孢杆菌等四种供试菌的抑菌活性及抑菌稳定性。采用试管半倍稀释法确定MIC和MBC,并探讨羟基酪醇对供试菌的生长和细胞膜完整性的影响以及在不同介质下的抑菌稳定性。结果表明,羟基酪醇对大肠杆菌、金黄色葡萄球菌、铜绿假单胞杆菌和枯草芽孢杆菌的MIC分别为0.625、0.625、1.250、2.500 mg/mL,MBC分别为1.250、1.250、2.500、5.000 mg/mL。与对照组相比,四种供试菌核酸和可溶性蛋白泄漏显著,细胞膜的完整性被破坏。在不同NaCl浓度下,羟基酪醇对枯草芽孢杆菌的抑菌活性稳定;在1.0%和2.0%NaCl浓度下,羟基酪醇对大肠杆菌和铜绿假单胞杆菌的抑菌活性稳定;在2.0%NaCl介质下低浓度的羟基酪醇对金黄色葡萄球菌的抑菌活性稳定,在0.5%、1.5%和2.0%NaCl介质下高浓度的羟基酪醇对金黄色葡萄球菌的抑菌活性稳定。在蔗糖介质中,羟基酪醇对四种供试菌的抑菌活性均不稳定。因此,羟基酪醇可以作为一种新型的防腐剂。     

Lipoic acid modified antimicrobial peptide with enhanced antimicrobial properties

RIWVIWRR-NH₂ (Bac8c) is a natural antimicrobial peptide (AMP) exhibiting great antibacterial activity against Gram-negative and Gram-positive bacteria. In this work, lipoic acid was used as a fatty acid hydrophobic ligand to modify Bac8c (LA-Bac8c) to further improve its antimicrobial properties. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assays showed that LA-Bac8c exhibited lower MIC (MBC) values against Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) than Bac8c. Similar results were reflected in the antibiofilm activity towards S. aureus and MRSA, and LA-Bac8c showed better activity to the biofilm which has been formed or is being formed. In addition to this, the obvious interaction between bacteria/biofilm and LA-Bac8c was observed by microscopy. LA-Bac8c displayed strong membrane depolarization and outer membrane permeabilizing ability, and the cell membrane treated with LA-Bac8c was destroyed to the leakage of bacteria cellular components. All these data indicated LA-Bac8c could be used as a useful antimicrobial peptide with wide application prospect.     

Antibacterial activities of temporin A analogs

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and -resistant Staphylococcus aureus as well as vancomycin-resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N-terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all-D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non-chiral interactions with membrane lipids.     

The cathelicidin-like peptide derived from panda genome is a potential antimicrobial peptide

A novel cathelicidin-like antimicrobial peptide was identified by mining genome of panda. This peptide (cathelicidin-AM) was synthesized. It showed potential antimicrobial activities against wide spectrum of microorganisms including Gram-negative and -positive bacteria, and fungi. It had similar antimicrobial abilities against both standard and clinically isolated drug-resistant strains. Cathelicidin-AM could rapidly exert its antibacterial activities. It just took less than 1 h to kill all Staphylococcus sciuri at the concentration of 2, 4 or 10 times of minimal inhibitory concentration (MIC) while clindamycin took 6 h. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis indicated that cathelicidin-AM killed bacteria by directly affecting bacterial cell wall and membrane. Phylogenetic analysis revealed that the panda cathelicidin had the nearest evolution relationship with dog cathelicidin. The current work provides a novel cathelicidin-like peptide with strong antimicrobial abilities.     

The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective alpha-helical IsCT-derived antimicrobial peptide

IsCT-P (ILKKIWKPIKKLF-NH2) is a novel alpha-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of L- or D-Pro kink on the structure and the mode of action of a short alpha-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT-p, in which D-Pro is substituted for L-Pro8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical alpha-helical structure in various membrane-mimicking conditions, whereas IsCT-p showed a random structure. This result indicated that D-Pro in the central position of a short alpha-helical peptide provides more remarkable structural flexibility than L-Pro. Despite its higher antibacterial activity, IsCT-p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT-p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT-p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central D-Pro kink in alpha-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.     

Proteome analyses of cellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone, a novel antibiotic candidate

The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC) values ranged from 31.25-62.5 μg/ml, and the minimal bactericidal concentration (MBC) was 250 μg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5-125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 μg/ml and 2 μg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 μg/ml) affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections.     

Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic

The growing public health problem of infections caused by multiresistant Gram-positive bacteria, in particular Staphylococcus aureus, prompted us to screen human epithelia for endogenous S. aureus-killing factors. A novel 5-kDa, nonhemolytic antimicrobial peptide (human beta-defensin-3, hBD-3) was isolated from human lesional psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes including multiresistant S. aureus and vancomycin-resistant Enterococcus faecium. Ultrastructural analyses of hBD-3-treated S. aureus revealed signs of cell wall perforation. Recombinant hBD-3 (expressed as a His-Tag-fusion protein in Escherichia coli) and chemically synthesized hBD-3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity and biochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD-3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supernatants revealed keratinocytes and airway epithelial cells as cellular sources of hBD-3. Tumor necrosis factor alpha and contact with bacteria were found to induce hBD-3 mRNA expression. hBD-3 therefore might be important in the innate epithelial defense of infections by various microorganisms seen in skin and lung, such as cystic fibrosis.