Using Bacteriophages to Prevent, Diagnose, and Treat Diseases

标签：Bacteriophage 细菌的两极表现出几种特殊的 与DNA和某些蛋白质的移动有关的功能。监测感染情况， 大肠杆菌细胞的光学显微镜，我们开发了 用量子点标记成熟噬菌体的程序。令人惊讶的是，大多数 发现感染的细菌附着在细菌杆上。这是真实的一些 温毒型E.广泛使用的大肠杆菌 不同的受体，并为细菌感染假结核耶尔森氏菌和霍乱弧菌。 感染细胞与极性蛋白标志物IcsAGFP共定位。许多，一个 e.大肠杆菌噬菌体λ DNA所需的蛋白质 注射，被发现定位细菌极以及。此外，lambda的标记 感染期间的DNA显示，它是在细胞的极区注射和复制的。 感染进化的好处导致了这种对极地的显著偏好 感染可能与λ的发育决定以及 在细菌细胞与环境交流的能力中， 调控噬菌体展示系统的研究 蛋白质-蛋白质相互作用 相互作用在蛋白质组学和药物发现中至关重要。2-Hybrid（2lambda） 系统仅限于体内环境。我们描述了一种 噬菌体2-研究蛋白质相互作用的杂交系统 体外诱饵和猎物以融合体的形式展示在λ噬菌体表面 它们被标记了不同的选择性耐药标记。一个互动的 在体外通过展示的蛋白质， 在极低的温度下，两种细菌感染导致双重耐药细菌菌落 多重感染。我们证明了蛋白分选信号泛素的相互作用 与Vps9-CUE，一种泛素结合结构域，以及通过（Gly-Glu）（4）和 （Gly-Arg）（4）肽。通过非融合（游离）诱饵或 猎物分子展示了我们的方法是多么强大和独特。我们还演示了使用 泛素和CUE展示结合蛋白，以在结合蛋白展示文库中找到结合伴侣。的 2lambda的独特用途也是 介绍了 使用细菌检测 量子点-纳米复合物与目前的关注， 随着耐药细菌和生物防御的发展，快速识别 传染性细菌传统的技术涉及分离和放大 病原菌是耗时的。我们报告了一种快速简单的方法， 工程化宿主特异性 噬菌体和噬菌体与链霉抗生物素蛋白包被的量子点的缀合。述的方法 在实验中提供每毫升少至10个细菌细胞的特异性检测 样品，在1小时内信号相对于背景放大约100倍。我们 相信该方法可以应用于任何对特定细菌敏感的细菌， 特别适用于检测生长缓慢的细菌菌株，例如， 分枝杆菌，或者是高度传染性的，例如，炭疽杆菌。的潜力 在单一样品中同时检测不同细菌种类及其在 讨论了噬菌体生物学的研究。

Summary: Bacteriophage infection The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsAGFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation. A phage display system to study protein-protein interactions Analyzing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro . Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described. Bacterial detection by the use of QD-nanocomplexes With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an approximately 100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.