Using Bacteriophages to Prevent, Diagnose, and Treat Diseases

摘要：噬菌体感染细菌的两极表现出几种与DNA和某些蛋白质的动员有关的特殊功能。为了用光学显微镜监测大肠杆菌细胞的感染，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，大多数感染噬菌体被发现附着在细菌杆上。这一点适用于使用截然不同受体的一些温带和强毒的大肠杆菌噬菌体，以及感染假结核耶尔森氏菌和霍乱弧菌的噬菌体。感染噬菌体与极性蛋白标记IcsAGFP共定位。许多，噬菌体lambda DNA注射所需的一种大肠杆菌蛋白，也被发现定位于细菌极。此外，在感染过程中对lambda DNA的标记表明，它是在感染的极区注射和复制的。导致这种对极地感染的显著偏好的进化优势可能与lambda的发育决定以及极点在细菌细胞与环境沟通能力和基因调控方面的功能有关。噬菌体展示系统用于研究蛋白质之间的相互作用，分析蛋白质之间的相互作用在蛋白质组学和药物发现中是至关重要的。2-杂交(2lambda)系统的使用仅限于体内环境。我们描述了一个用于体外研究蛋白质相互作用的噬菌体2-杂交系统。诱饵和猎物显示为与噬菌体Lambda表面的融合，这些噬菌体表面标记有不同的可选择抗药性标记。噬菌体在体外通过展示蛋白的相互作用允许细菌被两个噬菌体感染，从而在非常低的感染多样性下产生双重耐药细菌菌落。我们证明了蛋白质分类信号泛素与泛素结合区Vps9-CUE的相互作用，以及(Gly-Glu)(4)和(Gly-Arg)(4)肽的相互作用。非融合(游离)诱饵或猎物分子对噬菌体相互作用的干扰表明了我们的方法是多么的健壮和独特。我们还演示了使用泛素和提示展示噬菌体在lambda展示文库中寻找结合伙伴。还描述了2lambda的独特用途。利用QD-纳米复合体进行细菌检测结合目前人们对抗生素耐药细菌和生物防御的关注，对感染性细菌进行快速鉴定变得非常重要。涉及病原菌分离和扩增的传统技术非常耗时。我们报道了一种快速而简单的方法，它结合了工程宿主特异性噬菌体的体内生物素化和链霉亲和素包裹的量子点的偶联。该方法在实验样本中提供每毫升10个细菌细胞的特异性检测，在1小时内将背景信号放大约100倍。我们相信，该方法可应用于任何对特定噬菌体敏感的细菌，特别适用于检测生长缓慢的细菌菌株，如分枝杆菌，或高度传染性的细菌，如炭疽杆菌。讨论了在单个样品中同时检测不同细菌种类的可能性以及在噬菌体生物学研究中的应用。

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.