Extracellular DNA required for bacterial biofilm formation
Extracellular DNA required for bacterial biofilm formation
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DOI：
10.1126/science.295.5559.1487
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发表时间：
2002-02-22
期刊：
影响因子：
56.9
通讯作者：
Mattick, JS
中科院分区：
文献类型：
--
作者：
Whitchurch, CB;Tolker-Nielsen, T;Mattick, JS
细菌生物膜是封闭在自身产生的水合聚合物基质中的结构化细胞群落，其粘附于惰性或活性表面（1）。这些固着群落的形成及其对抗生素和宿主免疫攻击的固有抗性是许多持续性和慢性细菌感染的根源（1），包括由铜绿假单胞菌引起的感染，铜绿假单胞菌已作为生物膜形成的模型进行了深入研究（2，3）。将细菌生物膜保持在一起的基质是包括胞外多糖、蛋白质和DNA在内的大分子的复杂混合物（4）。后者被认为是来自裂解的细胞，并没有被认为是代表生物膜结构的重要组成部分。然而，多年来人们已经知道，一些细菌，包括铜绿假单胞菌，通过一种被认为与细胞裂解无关的机制产生大量的细胞外DNA，这种机制似乎涉及从外膜释放小泡（5，6）。在铜绿假单胞菌中藻酸盐生物合成的研究期间，我们发现在咔唑比色测定中反应的大多数细胞外物质不是胞外多糖而是DNA [如通过其在260 nm处的峰值吸光度、通过电泳显示和通过其脱氧核糖核酸酶（DNase）而不是核糖核酸酶敏感性所确定的]，因此假设该DNA可能在铜绿假单胞菌生物膜中起功能性作用。使用管环试验（2），我们发现向培养基中添加DNA酶I强烈抑制生物膜形成（Web图1A）（7），尽管不是细菌生长本身。然后，我们使用流动室系统更详细地研究了DNA酶I对生物膜形成的影响（8）。用绿色荧光蛋白（GFP）标记的铜绿假单胞菌PAO 1接种四个流动室通道，并分别用含有或不含DNA酶I的基本培养基冲洗两个通道。培养基中DNA酶I的存在防止了生物膜的形成。用不含DNA酶I的培养基冲洗的通道在3天后被广泛地定殖，而用含DNA酶I的培养基冲洗的通道在相同时期后基本上没有细胞，或含有很少的附着细胞（Web图1B）。为此，我们接种了五个流动室通道，并用不含DNA酶I的基本培养基冲洗它们，以允许建立不同年龄的铜绿假单胞菌生物膜。在不同的时间，将培养基转移到补充有DNA酶I的培养基，并且观察生物膜的命运。
Bacterial biofilms are structured communities of cells enclosed in self-produced hydrated polymeric matrix adherent to an inert or living surface (1). Formation of these sessile communities and their inherent resistance to antibiotics and host immune attack are at the root of many persistent and chronic bacterial infections (1), including those caused by Pseudomonas aeruginosa, which has been intensively studied as a model for biofilm formation (2, 3). The matrix, which holds bacterial biofilms together, is a complex mixture of macromolecules including exopolysaccharides, proteins, and DNA (4). The latter has been presumed to be derived from lysed cells and has not been thought to represent an important component of biofilm structure. However, it has been known for many years that some bacteria, including P. aeruginosa, produce substantial quantities of extracellular DNA through a mechanism that is thought to be independent of cellular lysis and that appears to involve the release of small vesicles from the outer membrane (5, 6). During studies of alginate biosynthesis in P. aeruginosa, we discovered that the majority of the extracellular material that reacted in the carbazole colorimetric assay was not exopolysaccharide but DNA [as determined by its peak absorbance at 260 nm, by electrophoretic display, and by its deoxyribonuclease (DNase) but not ribonuclease sensitivity] and therefore hypothesized that this DNA may play a functional role in P. aeruginosa biofilms. Using a tube ring assay (2), we found that addition of DNase I to the culture medium strongly inhibited biofilm formation (Web fig. 1A)(7), although not bacterial growth per se. We then investigated the effect of DNase I on biofilm formation in more detail using a flow-chamber system (8). Four flow-chamber channels were inoculated with green fluorescent protein (GFP)–tagged P. aeruginosa PAO1, and two channels each were irrigated with minimal medium with or without DNase I. The presence of DNase I in the medium prevented biofilm formation. The channels irrigated with medium without DNase I were extensively colonized after 3 days, whereas the channels irrigated with DNase I–containing medium were essentially without cells, or contained few attached cells, after the same period (Web fig. 1B).We also investigated whether DNase I could dissolve established biofilms. To this end, we inoculated five flow-chamber channels and irrigated them with minimal medium without DNase I to allow the establishment of P. aeruginosa biofilms of varying age. At various times, the medium was shifted to medium supplemented with DNase I, and the fate of the biofilms was