Bacteriophage in Prevention, Diagnosis and Treatment

几十年来，噬菌体如大肠杆菌噬菌体Lambda被广泛研究，作为研究基因调控、宿主-病毒相互作用和大分子组装等主题的模型系统。我们利用有关Lambda和相关噬菌体的信息库有两个目的： (1)诱变剂和致癌物的检测：我们开发了一种新的检测方法，用于检测小鼠体内Lambda转基因的突变。该方法使用突变(HFL)大肠杆菌选择只包含Lambda CII基因正向突变的噬菌体。除了直接选择相对容易之外，这种方法对自发和化学诱导突变的敏感性与广泛使用的突变靶基因lacI相当。此外，我们的检测成本比lacI系统低80倍。我们的CII检测系统现在正在全球范围内作为lacI系统的替代品使用。在与格伦·梅里诺博士的合作下，该系统现在正被用于研究突变在癌症发展中的作用。新基因的表达似乎导致了突变谱的变化。这个项目现在已经完成、发布和终止。 (2)噬菌体治疗：多重耐药细菌的流行促使我们尝试提高噬菌体的治疗效果。噬菌体作为抗菌剂的治疗应用受到哺乳动物宿主防御系统从循环系统中清除噬菌体颗粒的能力的阻碍。在我们关于细菌血症小鼠的研究中，为了减少宿主防御系统对噬菌体的清除，我们以前分离到了大肠杆菌噬菌体Lambda和沙门氏菌噬菌体P22突变体，它们能够在循环系统中停留更长时间，并在感染致命剂量细菌的动物中具有更强的抗菌能力。Lambda的突变发生在主要衣壳蛋白的基因中。在与卡尔·梅里尔博士和迪恩·肖尔博士的合作下，我们继续了我们的噬菌体治疗项目，通过分离和鉴定合适的噬菌体来治疗其他致病性大肠杆菌菌株。一种强毒的双链DNA噬菌体f K1-5已经被分离出来，并被发现能够感染具有K1或K5多糖壳的致病性大肠杆菌菌株。电子显微镜显示，该病毒粒子由一个小的二十面体头部和短的尾尖组成，与泊多病毒科的成员相似。对尾纤维蛋白编码区的DNA序列分析显示，有两个开放阅读框编码先前特征的水解噬菌体尾纤维蛋白。这是Phi K5的K5裂解酶蛋白基因中的第一个，它允许该噬菌体特异性地感染K5大肠杆菌菌株。第二个开放阅读框编码的蛋白质在氨基酸序列上与Phi K1E的N-乙酰神经氨酸酶(内唾液酸酶)蛋白几乎相同，这使得该噬菌体能够特异性地感染大肠杆菌的K1株。我们提供了成熟的噬菌体颗粒含有两种尾纤维蛋白的实验证据，突变分析表明每种蛋白都可以独立失活。Phi K5、Phi K1E和Phi K1-5的尾部基因序列比较表明，这些基因呈模块化或盒状排列，表明该噬菌体家族可以通过水平基因转移扩大寄主范围。

Bacteriophages such as the Escherichia coli phage Lambda have been studied extensively for several decades as model systems for the study of such topics as gene regulation, host-virus interactions, and macromolecular assembly. We have taken advantage of the base of information about Lambda and related phages for two purposes: (1) Detection of mutagens and carcinogens: We developed a novel assay for the detection of mutations in a Lambda transgene contained in mice. The assay selects for phage containing forward mutations only in the Lambda cII gene, using a mutant (hfl) Escherichia colihost. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutation was comparable to the widely used mutational target gene lacI. Moreover, our assay costs 80 times less to use than the lacIsystem. Our cII assay system is now being used worldwide as a replacement for the lacIsystem. In a collaboration with Dr. Glenn Merlino, the system is now being used to study the role of mutagenesis in cancer development. It appears that expression of the new gene causes a change in the spectrum of mutations. This project has now been completed, published, and terminated. (2) Bacteriophage therapy: The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by the capacity of mammalian host defense systems to remove phage particles from the circulatory system. In our studies involving bacteremic mice, to reduce phage elimination by the host defense system, we previously isolated E. coli phage Lambda and Salmonella phage P22 mutants able to remain in the circulatory system for longer periods of time and also have greater capability as antibaceterial agents in animals infected with lethal doses of bacteria. The mutations in Lambda are in the gene for the major capsid protein. In a collaboration with Dr. Carl Merril and Dr. Dean Scholl, we have continued our phage therapy project with other pathogenic E. coli strains by isolating and characterizing suitable phages. A virulent double-stranded DNA bacteriophage, f K1-5, has been isolated and found to be capable of infecting pathogenic Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs showed that the virion consists of a small icosohedral head with short tail spikes, similar to members of the Podoviridae family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first in the K5 lyase protein gene of Phi K5, which allows this phage to specifically infect K5 E. coli strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of Phi K1E, which allows this phage to specifically infect K1 strains of E. Coli. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of Phi K5, Phi K1E, and Phi K1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer.