海洋芽孢杆菌源脂肽CLPs抑制溶藻弧菌运动性分子机制研究

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	海洋芽孢杆菌源脂肽CLPs抑制溶藻弧菌运动性分子机制研究
	修鹏远
学位类型	硕士
导师	孙超岷
	2017-05-17
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	脂肽 Pumilacidin 溶藻弧菌运动性抗生物膜
其他摘要	运动性对于病原菌成功入侵宿主、定殖、并在宿主体内释放毒力因子具有重要作用，因此细菌运动性是一个新型药物筛选潜在的靶标。在本研究中，从雅浦海沟附近海域分离到了一株海洋溶藻弧菌Vibrio alginolyticus 178，该菌具有强烈的运动能力且对斑马鱼有高致病性。以溶藻弧菌V. alginolyticus 178作为对象，利用对峙培养法从相同生态位筛选得到一株抑制溶藻弧菌V. alginolyticus 178运动的海洋细菌芽孢杆菌Bacillus sp. 176。采用盐酸沉淀、凝胶过滤层析、高效液相色谱等方法从芽孢杆菌Bacillus sp. 176的发酵液中分离得到了两个活性物质（CLP1和CLP2），运用氨基酸分析、质谱和核磁共振波谱的技术方法鉴定了这两个活性物质的结构。这两个活性物质是pumilacidin类的脂肽，分子量在脂肪链上相差一个亚甲基。除了能够抑制溶藻弧菌V. alginolyticus 178的运动外，活性脂肽（CLPs）能够明显降低溶藻弧菌对斑马鱼的致病性，诱导细菌细胞的聚集沉降，并且降低溶藻弧菌V. alginolyticus 178在玻片上的黏附能力。此外，活性脂肽能够抑制其它病原菌生物膜的形成而不杀死病原菌。qRT-PCR实验结果表明，经过脂肽处理后，溶藻弧菌V. alginolyticus 178的两个鞭毛合成组装基因（flgA 和flgP）转录水平显著下调。本研究表明芽孢杆菌Bacillus sp. 176产生的活性脂肽能够抑制溶藻弧菌的运动性，具有作为新型抗菌先导化合物的潜力，靶向细菌的运动性和生物膜的形成而不产生抗生素耐性。; Bacterial motility is crucial for the successful infection, colonization, and virulence in hosts by pathogens. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. A marine bacterium Vibrio alginolyticus 178 was isolated from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. According to screening assays, we found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium Bacillus sp. 176 isolated from the same niche. Two different active substances (CLPs) were isolated and purified from Bacillus sp. 176 using acid precipitation, gel filtration chromatography, and high performance liquid chromatography. The structures of active substances were characterized by amino acid analysis, mass spectrometry and nuclear magnetic resonance spectroscopy. The two active substances (CLPs) both have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs could effectively reduce the pathogenicity of V. alginolyticus 178, induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLPs treatment the transcription levels of two V. alginolyticus flagella assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. These findings suggest that CLPs from Bacillus sp. 176 show great promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136563
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	修鹏远. 海洋芽孢杆菌源脂肽CLPs抑制溶藻弧菌运动性分子机制研究[D]. 北京. 中国科学院大学,2017.

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