厚壳贻贝两种新型防御素的分子鉴定

贻贝是全球范围内具有重要经济价值和生态价值的双壳贝类。贻贝抗菌肽具有极强的分子多样性,也是当前抗菌肽研究的重要对象。防御素是贻贝抗菌肽的重要成员, 从厚壳贻贝中鉴定到2种新型防御素, 但其分子特性和免疫机制尚不清楚。为此, 对厚壳贻贝体内新发现的2种防御素开展研究。序列分析结果表明,2种新型防御素均具有节肢动物防御素结构特征,因而被命名为arthropod like defensin (ALD)。利用荧光定量PCR研究了2种防御素在贻贝不同组织及不同发育阶段的表达量差异。进一步分析了2种防御素在3种不同微生物诱导下的表达量时间曲线。利用固相化学合成技术对2种防御素的成熟肽区进行合成并开展了功能验证。研究结果表明, 2种ALD 主要表达部位在外套膜和消化腺, 且ALD-1具有雄性特异表达特征。此外, ALD-1和ALD-2在贻贝幼虫阶段均未表达; 在不同微生物刺激下, 2种ALD表现出不同的免疫反应模式, 显示出2种防御素具有不同的免疫调节机制。化学合成的2种ALD均具有抑菌活性, 其对不同微生物的抑制率在20%~80%之间。上述研究为深入了解贻贝免疫防御的分子机制,以及贻贝抗菌肽的免疫功能和后续的分子资源开发奠定了基础。     

一种新型贻贝抗菌肽的分离纯化及鉴定

厚壳贻贝（Mytilus coruscus）广泛分布于我国东部海域,其体内富含各种抗菌肽分子,是研究软体动物免疫防御机制以及开发抗菌肽来源的新型生物抗生素的重要对象。采用多步反相高效液相色谱对厚壳贻贝血清进行分离纯化,获得一种分子量为6261.55 D的具有抗菌活性的多肽成分;经多肽N端测序和基因克隆,结果表明该抗菌肽由55个氨基酸残基构成,含6个半胱氨酸并形成三对二硫键。结构域分析表明该抗菌肽具有几丁质结合结构域（Chitin-biding domain）,因此将该抗菌肽命名为mytichitin-A。Mytichitin-A对革兰氏阳性菌具有较强的抑制作用,同时对真菌及革兰氏阴性菌也具有抑制作用。荧光定量PCR检测表明,mytichitin-A主要在厚壳贻贝的性腺组织中表达且在细菌诱导后12h其表达量达到峰值。研究为深入了解厚壳贻贝抗菌肽的分子多样性及免疫机制奠定了基础。       

两种合成抗菌肽的结构及抗菌作用机理

为深入了解两种新型人工抗菌肽mytilin-derived-peptide-1 (MDP-1) 和mytilin-derived-peptide-2 (MDP-2) 的溶液结构和抗菌机理并探讨两种抗菌肽之间活性差异的结构基础,采用二维核磁共振技术 (2-D NMR) 研究MDP分子的溶液结构;采用透射电镜技术 (Transmitted electron microscopy,TEM) 研究MDP分子对于大肠杆菌和藤黄叠球菌的作用机理。研究结果表明,MDP-1和MDP-2均采取了典型的β-发夹结构,其分子表面具有明显的疏水斑片,其分子中碱性氨基酸突出于分子表面;经MDP分子处理后的大肠杆菌以及藤黄叠球菌均出现细胞壁或细胞膜结构被破坏,并出现膜壁分离以及细胞质内缩现象。我们认为,MDP-1和MDP-2分子中的碱性氨基酸有助于MDP结合细菌表面的带负电荷的基团,同时其分子表面的疏水斑片有助于其插入到细菌细胞膜内;其疏水斑片面积以及碱性氨基酸在分子表面的拓扑结构差异是MDP-1和MDP-2活性差异的主要原因;电镜实验结果表明MDP-1和MDP-2的主要靶标是细菌细胞壁以及细胞膜;上述研究为深入了解MDP分子的结构与功能的关系以及将来基于MDP分子的药物研发奠定了基础。     

厚壳贻贝足丝盘黏附蛋白mcofp-3的重组真核表达

厚壳贻贝(Mytilus coruscus)黏附蛋白分子mcofp-3(M.coruscusfoot protein-3)主要分布于贻贝足丝盘,贻贝在水环境下的黏附过程中起到关键作用,但因其难溶于水且在贻贝足丝盘中含量极低,故妨碍了对其进行深入研究。为建立厚壳贻贝足丝蛋白mcofp-3的真核表达体系,并获得足够的mcofp-3黏附蛋白进行后续研究,采用酵母表达体系对mcofp-3进行了重组表达。通过PCR方法克隆厚壳贻贝的mcofp-3基因,构建mcofp-3的酵母真核表达载体pVT102U/α/mcofp-3,鉴定结果表明,重组表达质粒pVT102U/α/mcofp-3由真核载体pVT102U/α和mcofp-3的成熟肽DNA片段组成,插入的mcofp-3成熟肽DNA片段与预期序列完全一致;采用LiAC转化法将重组表达质粒转化到S78酿酒酵母中,经过RT-PCR分析以及1.0%的琼脂糖凝胶电泳检测,结果表明,重组的mcofp-3得到了成功的转录;发酵菌液经阳离子交换柱及高效液相色谱分离,以及Tris-Tricine-SDS-PAGE检测,结果表明,重组的厚壳贻贝黏附蛋白分子mcofp-3得到了成功表达,表达...     

厚壳贻贝新型抗菌肽myticalin和mytimacin的分子鉴定

从厚壳贻贝(Mytilus coruscus)血细胞转录组数据中鉴定到两种新型抗菌肽, 分别为myticalin和mytimacin。为了解两种抗菌肽的结构与功能, 以及在贻贝免疫过程中的响应模式, 采用固相化学合成技术获得两种抗菌肽化合物, 在此基础上开展了抑菌活性测试, 红细胞毒性测试及对微生物抑制作用机理的扫描电镜观察。此外, 研究了贻贝在不同微生物诱导下, 两种抗菌肽的表达模式。研究结果表明, 化学合成的myticalin和mytimacin均具有抑菌活性, 但抑菌谱有所差异。两种抗菌肽尽管结构差异较大, 但对金黄葡萄球菌和溶藻弧菌的作用机制类似, 均能导致细菌表面形态结构发生变化。此外, mytimacin对白色念珠菌表现出明显抑制作用, 且其作用机制不同于金黄葡萄球菌和溶藻弧菌, 能导致白色念珠菌表面出现孔洞, 而myticalin则无此现象。两种抗菌肽在不同微生物诱导后, 其表达量均明显上调, 但myticalin表现出对革兰氏阳性菌诱导的敏感性, 而mytimacin表现出对真菌和革兰氏阳性菌诱导的敏感性。研究为深入了解贻贝抗菌肽的分子多样性及其抑菌活性机制, 以及贻贝抗菌肽的分子工程研究奠定了基础。     

厚壳贻贝Mytilin-1成熟肽在毕赤酵母中的重组表达及其抑菌活性

贻贝素(Mytilins)是一类主要在贝类血细胞中表达的阳离子小分子抗菌肽,包括多种同工型,它们在免疫系统中发挥了重要作用,Mytilin-1是其中的一种同工型。厚壳贻贝(Mytilus coruscus)Mytilin-1的一级结构由信号肽、成熟肽和C端的延伸肽组成;其成熟肽决定了Mytilin-1的生物学活性,它由34个氨基酸残基组成,其中8个保守的半胱氨酸残基在空间上能形成4对二硫键,对Mytilin-1的稳定性和活性起了关键作用。以厚壳贻贝的Mytilin-1成熟肽为重组DNA表达的目的蛋白,根据毕赤酵母(Pichia pastoris)的密码子偏爱性对编码该成熟肽的密码子进行优化,合成的目的基因"mMy1"与表达载体pPICZαA连接后电转至毕赤酵母X-33,通过高浓度博来霉素筛选高拷贝酵母转化子,获得的阳性转化子在29℃、250 r/min、pH6.0的条件下,使用1%甲醇诱导表达96 h;培养液上清经Tricine-SDS-PAGE和Western blot分析证明:重组mMy1在毕赤酵母X-33中得到成功表达,其分子量为4.8 kD。抑菌试验表明,重组mMy1对革兰氏阳性的金黄色葡萄球菌(Staphylococcus aureus)和枯草芽孢杆菌(Bacillus subtilis)以及革兰氏阴性的大肠杆菌(Escherchia coli)具有抑菌活性。     

基于illumina测序的厚壳贻贝足组织转录组研究

贻贝足丝是贻贝足组织分泌的足丝蛋白形成的非细胞组织,具有在水环境下的极强粘附性能,是当前生物粘附剂及抗腐蚀材料的研发热点．为进一步了解贻贝足丝蛋白的分子多样性特征,采用新一代Illumina高通量测序平台对厚壳贻贝(Mytilus coruscus)足组织进行转录组测序,首次构建了厚壳贻贝足组织的转录组数据库．共计获得7 199 799 840 nt的碱基数据经过序列拼接和组装,获得88 825条unigene．对上述unigene开展了序列注释,共计37 007条unigene获得注释．在此基础上,经序列检索和比对,从中筛选出与目前已知的11种足丝蛋白同源的56条unigene序列并进行分析．结果表明,厚壳贻贝足丝蛋白具有明显的氨基酸偏好性,部分足丝蛋白具有重复序列,且厚壳贻贝足丝蛋白与其他种类的贻贝足丝蛋白具有较高的序列相似性．上述结果为后续贻贝足丝蛋白的批量鉴定以及在此基础上的贻贝足丝形成、固化以及粘附机制相关研究奠定了基础．     

鱼类抗菌肽的研究进展

鱼类抗菌肽是鱼体天然免疫的重要组成部分,其结构和组成复杂多样.根据生化和结构特点,可将它们分为4种基本类型：具有疏水或双亲性α螺旋结构的抗菌肽、含多对二硫键并可形成β折叠结构的抗菌肽、组蛋白样抗菌肽和具有糖基化修饰的抗菌肽.鱼类抗菌肽多以前肽原的形式合成,通过酶解切除信号肽和羧基端酸性片段后形成有活性的成熟肽.成熟肽具有很强的抑菌活性,其最小抑制浓度多在毫摩尔水平.目前,已克隆了多个鱼类抗菌肽基因,揭示了pleurocidin等基因家族的结构及其转录调控特点.鱼类抗菌肽的抗菌机制已建立了“桶-桶板”和“地毯”样两种模型,基本阐明了抗菌肽分子结构与抗菌功能间的关系.     

利用蛋白质组学手段鉴定新型贻贝足丝蛋白

贻贝通过足腺分泌特有的足丝并以此粘附于水下各种基质表面.贻贝足丝中富含各种粘附蛋白,其优异的水下粘附性能使其成为开发新型生物粘合剂的候选分子.厚壳贻贝足丝粘附能力强,本文采用尿素及盐酸胍抽提结合二维双向电泳技术（two-dimensional electrophoresis, 2-DE）,分别对厚壳贻贝足丝纤维和足丝盘的蛋白质进行分离及染色;采用串联质谱技术结合常规搜库和表达序列标签（EST) 数据库搜索,对分离获得的蛋白质点进行鉴定,从中获得了mfp-3、mfp-6、胶原蛋白以及3种未曾报道过的新型贻贝足丝蛋白成分.上述研究为深入了解厚壳贻贝足丝蛋白的分子多样性、探讨其粘附机理以及从中筛选具有应用前景的贻贝足丝蛋白奠定了基础.     

厚壳贻贝低分子量足丝蛋白的初步鉴定(英文)

足丝蛋白是贻贝科(Mytilidae)所特有一种在水环境中也能表现出强黏附功能的蛋白,也是目前开发新型生物黏附剂的主要候选分子。厚壳贻贝(Mytilus coruscus)广泛分布于我国东部沿海,是我国具有重要经济价值的贻贝,其足丝粗硬,黏附力强,关于厚壳贻贝的足丝蛋白的研究目前尚未见报道。通过醋酸抽提结合反相高效液相色谱分离,从厚壳贻贝足丝盘中分离纯化到数种低分子量足丝蛋白,经质谱鉴定和氨基酸序列测定,其中三种足丝蛋白(分子量6 kD左右)属于贻贝足丝蛋白-3(mytilus foot protein-3,mfp-3)家族,且序列中富含DOPA,另有三种足丝蛋白为未知新型足丝蛋白。石英晶体微天平分析表明,厚壳贻贝低分子量足丝蛋白在金表面有较强的吸附能力,这与其黏附功能是直接相关的。以上工作为深入了解厚壳贻贝低分子量足丝蛋白的分子多样性以及黏附机制奠定了基础。     

Lipopolysaccharide induces amyloid formation of antimicrobial peptide HAL-2

Lipopolysaccharide (LPS), the important component of the outer membrane of Gram-negative bacteria, contributes to the integrity of the outer membrane and protects the cell against bactericidal agents, including antimicrobial peptides. However, the mechanisms of interaction between antimicrobial peptides and LPS are not clearly understood. Halictines-2 (HAL-2), one of the novel antimicrobial peptides, was isolated from the venom of the eusocial bee Halictus sexcinctus. HAL-2 has exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria and even against cancer cells. Here, we studied the interactions between HAL-2 and LPS to elucidate the antibacterial mechanism of HAL-2 in vitro. Our results show that HAL-2 adopts a significant degree of β-strand structure in the presence of LPS. LPS is capable of inducing HAL-2 amyloid formation, which may play a vital role in its antimicrobial activity.     

The characteristic region of arenicin-1 involved with a bacterial membrane targeting mechanism

The antimicrobial peptide arenicin-1 consists of two antiparallel β-sheets linked by a hydrophilic β-turn. To determine the role of a specific region found in a particular β-sheet structure of the peptide for antibacterial activity, two analogs with N-terminal deletions (RW) and substitutions of Arg to Ala in the β-turn region were designed. In the minimum inhibitory concentration (MIC) test, the antibacterial activities of the analogs were reduced for both Gram-positive and Gram-negative bacteria, when compared to arenicin-1. The influence of the decrease in hydrophobicity on the antibacterial activity was confirmed by a hemolytic assay. Through flow cytometric analysis using propidium iodide (PI) and a 1,6-diphenyl-1,3,5-hexatriene (DPH) assay, it was confirmed that the analogs decreased the degree of plasma membrane permeability compared to arenicin-1. In particular, analog 2 showed a lower permeability in Gram-negative bacteria than in Gram-positive bacteria. The results indicate that a reduction in the net charge weakened the electrostatic interactions between the peptides and the negatively charged membranes. In liposomes, which mimic bacterial membranes, due to a reduced binding affinity to the membranes, the analogs could not deeply penetrate into the hydrocarbon region and induce enough fluorescein isothiocyanate-dextran (FD) leakage compared to that of arenicin-1. It is thought that the Arg residue in the hydrophilic β-turn region is more important to antibacterial activity than the Arg residue in the N-terminal region. This study suggests that the Arg and Trp residues in the N-terminal region and the Arg residue in the β-turn region of arenicin-1 play a key role in antibacterial activity.     

Human peptidoglycan recognition proteins require zinc to kill both gram-positive and gram-negative bacteria and are synergistic with antibacterial peptides

Mammals have four peptidoglycan recognition proteins (PGRPs or PGLYRPs), which are secreted innate immunity pattern recognition molecules with effector functions. In this study, we demonstrate that human PGLYRP-1, PGLYRP-3, PGLYRP-4, and PGLYRP-3:4 have Zn(2+)-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria at physiologic Zn(2+) concentrations found in serum, sweat, saliva, and other body fluids. The requirement for Zn(2+) can only be partially replaced by Ca(2+) for killing of Gram-positive bacteria but not for killing of Gram-negative bacteria. The bactericidal activity of PGLYRPs is salt insensitive and requires N-glycosylation of PGLYRPs. The LD(99) of PGLYRPs for Gram-positive and Gram-negative bacteria is 0.3-1.7 muM, and killing of bacteria by PGLYRPs, in contrast to killing by antibacterial peptides, does not involve permeabilization of cytoplasmic membrane. PGLYRPs and antibacterial peptides (phospholipase A(2), alpha- and beta-defensins, and bactericidal permeability-increasing protein), at subbactericidal concentrations, synergistically kill Gram-positive and Gram-negative bacteria. These results demonstrate that PGLYRPs are a novel class of recognition and effector molecules with broad Zn(2+)-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria that are synergistic with antibacterial peptides.     

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.     

Antimicrobial activities of heparin-binding peptides.

Antimicrobial peptides are effector molecules of the innate immune system. We recently showed that the human antimicrobial peptides alpha-defensin and LL-37 bind to glycosaminoglycans (heparin and dermatan sulphate). Here we demonstrate the obverse, i.e. structural motifs associated with heparin affinity (cationicity, amphipaticity, and consensus regions) may confer antimicrobial properties to a given peptide. Thus, heparin-binding peptides derived from laminin isoforms, von Willebrand factor, vitronectin, protein C inhibitor, and fibronectin, exerted antimicrobial activities against Gram-positive and Gram-negative bacteria. Similar results were obtained using heparin-binding peptides derived from complement factor C3 as well as consensus sequences for heparin-binding (Cardin and Weintraub motifs). These sequence motifs, and additional peptides, also killed the fungus Candida albicans. These data will have implications for the search for novel antimicrobial peptides and utilization of heparin-protein interactions should be helpful in the identification and purification of novel antimicrobial peptides from complex biological mixtures. Finally, consensus regions may serve as templates for de novo synthesis of novel antimicrobial molecules.     

Interactions of two enantiomers of a designer antimicrobial peptide with structural components of the bacterial cell envelope

Antimicrobial peptides (AMPs) have great potential in treating multi-drug resistant bacterial infections. The antimicrobial activity of d -enantiomers is significantly higher than l -enantiomers and sometimes selectively enhanced against Gram-positive bacteria. Unlike phospholipids in the bacterial plasma membrane, the role of other bacterial cell envelop components is often overlooked in the mode of action of AMPs. In this work, we explored the structural interactions between the main different structural components in Gram-negative/Gram-positive bacteria and the two enantiomers of a designer AMP, GL13K. We observed that both l -GL13K and d -GL13K formed self-assembled amyloid-like nanofibrils when the peptides interacted with lipopolysaccharide and lipoteichoic acid, components of the outer membrane of Gram-negative bacteria and cell wall of Gram-positive bacteria, respectively. Another cell wall component, peptidoglycan, showed strong interactions exclusively with d -GL13K and formed distinct laminar structures. This specific interaction between peptidoglycans and d -GL13K might contribute to the enhanced activity of d -GL13K against Gram-positive bacteria as they have a much thicker peptidoglycan layer than Gram-negative bacteria. A better understanding of the specific role of bacterial cell envelop components in the AMPs mechanism of action can guide the design of more effective Gram-selective AMPs.     

RYH: a minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity

Bovine hemoglobin is an animal protein described as source of bioactive peptides. Enzymatic hydrolysis of this protein results into some peptides exhibiting antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, a family of peptides from the beta chain (beta-114-145 derived peptides) obtained by peptic hydrolysis of bovine hemoglobin, was purified by reverse-phase HPLC and characterized by different analytical techniques (mass spectrometry, circular dichroism). The minimum inhibitory concentration was determined to show the antimicrobial activity of these peptides. Four bacterial strains were used: two Gram-negative (Escherichia coli and Salmonella Enteritidis) and two Gram-positive strains (Listeria innocua and Micrococcus luteus). The effect of these peptides on artificial membrane was also measured. Our findings showed that the peptide β114-145 and its peptic derivatives contain the RYH sequence. The most antimicrobial peptide is the RYH peptide which was the shortest one.     

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.