Bacteriophage in Prevention, Diagnosis and Treatment

噬菌体如大肠杆菌噬菌体λ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已经被广泛研究了几十年。我们出于两个目的利用了关于Lambda和相关知识库的信息： (1)诱变剂和致癌物的检测：我们开发了一种用于检测小鼠中所含λ转基因突变的新方法。该试验使用突变型（hfl）大肠杆菌宿主选择仅在λ cII基因中含有正向突变的噬菌体。除了相对容易的直接选择，自发和化学诱导的突变的灵敏度，这种检测方法是广泛使用的突变靶基因lacI。此外，我们的检测成本比lacI系统低80倍。我们的cII检测系统目前正在全球范围内使用，作为lacI系统的替代品。在与Glenn梅尔利诺博士的合作中，该系统现在被用于研究突变在癌症发展中的作用。新基因的表达似乎引起了突变谱的变化。该项目现已完成、发布并终止。 (2)噬菌体疗法：多重耐药细菌病原体的流行率增加促使我们尝试提高噬菌体的治疗效果。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阻碍了细菌抗菌剂的治疗应用。在我们对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，我们先前分离了E。大肠杆菌噬菌体λ和沙门氏菌噬菌体P22突变体能够在循环系统中保留更长的时间，并且在被致死剂量的细菌感染的动物中也具有更大的作为抗菌剂的能力。λ的突变是在主要衣壳蛋白的基因中。在与Carl Merril博士和Dean Scholl博士的合作中，我们继续了我们对其他致病性大肠杆菌的噬菌体治疗项目。大肠杆菌菌株分离和表征合适的大肠杆菌。已分离出一种毒性双链DNA噬菌体f K1-5，并发现其能够感染具有K1或K5多糖荚膜的致病性大肠杆菌菌株。电子显微照片显示，病毒粒子由一个小的二十面体头部和短尾刺组成，类似于短尾病毒科的成员。编码尾丝蛋白的区域的DNA序列分析显示两个开放阅读框编码先前表征的水解噬菌体尾丝蛋白。第一个在K5裂解酶蛋白基因的Phi K5，这使得该噬菌体特异性感染K5 E。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 K1 E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感染大肠杆菌K1菌株。杆菌我们提供的实验证据表明，成熟的噬菌体颗粒包含两个尾纤维蛋白，和突变分析表明，每个蛋白质可以独立地失活。Phi K5、Phi K1 E和Phi K1-5的尾部基因区的比较显示，基因以模块或盒构型排列，并表明该家族的Escherichia coli可以通过水平基因转移来扩大宿主范围。

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.