The Use of Bacteriophage in the Prevention, Diagnosis, and Treatment of Human Di

< p > 1。噬菌体疗法。在过去，我们一直在开发噬菌体，用于治疗食物、动物和人类中的几种细菌感染。我们开发了几个这样的噬菌体，如下所述。我们选择了特异性噬菌体来治疗(i)万古霉素耐药<i>粪肠球菌</i>感染，(ii) <i>E。大肠杆菌</i> K1和K-5感染，沙门氏菌</i>感染。我们将每个噬菌体描述为毒力型（非溶原性），不携带任何毒素基因，在短时间内具有高爆发大小（如< 1 >E）。大肠杆菌</i>噬菌体T7)，具有广泛的宿主特异性。具体来说，VRE噬菌体为phiENB6， < 1 >E噬菌体为phiK-7和phiK1-5。大肠杆菌</i>， phiSP6和新分离的噬菌体phi111用于食源性<i>沙门氏菌</i>致病菌。除了<i>沙门氏菌</i>噬菌体phi111外，近4年来在该领域完成的所有研究，包括用实验菌拯救动物的实验都已发表。目前，在韩国首尔国立大学（Seoul National University）工作的sang - yeol Ryu博士正在进行涉及沙门氏菌phil111 <i> <i>的动物实验。尽管我们成功地分离或鉴定和表征了适当的噬菌体，并成功地将其用于拯救实验菌群中的动物，但我们已经无限期地推迟了我们自己将项目推向临床水平的努力。在过去，这些试验是由两家噬菌体技术公司（指数生物疗法公司和Gangagen公司）在与我们协商后进行的。然而，由于最近的NIH指南不鼓励商业企业继续与我们互动，我们不能继续这种共同努力。尽管如此，我们扩大了对开发工程噬菌体的兴趣，以便在临床和环境样品中轻松检测特定细菌。我们还成功地开发了使用噬菌体lambda作为蛋白质展示剂的方法。此外，我们利用lambda-display噬菌体系统构建了一个蛋白- 2杂交系统，称为2lambda系统，研究蛋白-蛋白在体外</i> </i>的相互作用。现将近四年来噬菌体技术的研究成果总结如下：(i)在哺乳动物循环系统中存活的λ噬菌体突变体的表征。在无菌小鼠实验中，不含噬菌体lambda的适应性抗体，在腹腔静脉或口服给药48小时内，循环系统中噬菌体滴度下降超过10(9)pfu。基于这些观察，我们之前使用连续传代技术选择lambda噬菌体突变体，在腹腔注射后24小时在小鼠循环系统中的容量增加了13,000-16,000倍。这些长时间循环的噬菌体在原始分离株中至少有三个突变。我们现在已经证明，其中一种变化发生在主要的噬菌体衣壳(E)蛋白中，它导致谷氨酸在残基158处变为赖氨酸，并足以赋予长循环表型，这可能是逃避先天免疫的原因。(二)。<i>沙门氏菌</i>和<i>的鉴定大肠杆菌</i>噬菌体的治疗价值。我们已经确定了两个溶解噬菌体的基因组序列，SP6 phi111，感染<i>鼠伤寒沙门氏菌</i> LT2，和phiK1-5，感染<i>E。大肠杆菌</i>血清型K1和K5。除phi111外，噬菌体的基因组组织几乎相同，但尾巴纤维基因具有不同的宿主特异性。SP6和phiK1-5似乎在核苷酸水平上有广泛的差异，但它们之间的关系仍然比目前已知的任何其他噬菌体更密切。SP6和K1-5基因组分别含有43,769 bp和44,385 bp，直接末端重复序列分别为174 bp和234 bp。在两个基因组的105个假定开放阅读框中，约有一半与数据库中已知或预测的功能明显有利于噬菌体生长的蛋白质没有显著的相似性。SP6和K1-5的整体基因组组织与T7噬菌体相当，尽管编码DNA代谢功能的基因的具体顺序尚未保守。（iii）荚膜多糖可能是噬菌体入侵的屏障。<我> E。产生K1多糖胶囊的大肠杆菌</i>菌株长期以来与发病机制有关。这种胶囊被认为可以增加细胞的侵袭性，使细菌避免被补体吞噬和失活。它也被一些噬菌体识别为受体，如K1F和K1-5，它们具有降解多糖的病毒粒子相关酶。我们已经证明K1囊在< 1 >E的表达。大肠杆菌</p>通过T7物理阻断感染，T7是一种识别脂多糖为主要受体的噬菌体。利用噬菌体和量子点进行高灵敏度的细菌检测。随着目前对抗生素耐药细菌和生物防御的关注，快速识别感染性细菌变得非常重要。我们开发了一种快速简便的方法，结合了工程宿主特异性噬菌体<i>在体内</i>的生物素化，并将噬菌体与链霉亲和素包被的量子点结合。该方法在实验和环境样品中提供每毫升10个或更少的细菌细胞的特异性检测，在一小时内将背景信号放大约100倍。由于可以用不同颜色的量子点标记不同的噬菌体，因此在单个样品中同时检测不同细菌种类和在噬菌体生物学研究中的应用现在是可行的。</p><p>3。一个基于lambda的遗传系统来研究蛋白-蛋白相互作用<i>在体外</i>: 2lambda系统。分析蛋白质-蛋白质相互作用在蛋白质组学和药物发现中至关重要。2-Hybrid系统的使用仅限于<i /i / >的体内环境。我们开发了lambda 2-Hybrid系统，用于研究蛋白质相互作用<i> </i>。诱饵和猎物被显示为噬菌体λ表面蛋白D的融合体，并标记有不同的可选择的耐药标记。噬菌体在体外</i> </i>通过所显示的蛋白相互作用，允许细菌被两个噬菌体感染，产生溶原细胞，在非常低的感染率下，在双耐药细菌菌落上检测到溶原细胞，在这种感染率下，巧合的双重感染几乎为零。我们证明了蛋白质分选信号泛素与泛素结合域Vps9-CUE以及[Gly-Glu]4和[Gly-Arg]4肽的相互作用。非融合（自由）诱饵或猎物分子对噬菌体相互作用的中断表明，我们的方法是多么强大和独特。我们还展示了使用Ubiquitin和CUE展示噬菌体在lambda-display库中寻找结合伙伴

<p>1. Bacteriophage therapy. In the past, we have been developing bacteriophages for their potential uses in the treatment of several bacterial infections in food, animals and humans. We developed several such phages as detailed below. We selected specific phages to treat (i) vancomycin resistant <i>Enterococeus faecalis</i> infections, (ii) <i>E. coli</i> K1 and K-5 infections, and (iii) <i>Salmonella</i> infections. We characterized each phage to be of virulent type (non-lysogenizing), does not carry any toxin genes, gives high burst size within a short time (like <i>E. coli</i> phage T7) and have broad host specificity. Specifically, the phages are phiENB6 for VRE, phiK-7 and phiK1-5 for <i>E. coli</i>, phiSP6 and a newly isolated phage phi111 for food borne <i>Salmonella</i> pathogens. Except for the <i>Salmonella</i> phage phi111, all of the research accomplished in this area during the last 4 years, including the experiments to show the rescue of animals with experimental bacterimia have been published. The animal experiments involving phi111 of <i>Salmonella</i> is now being carried out, by Dr. Sangryeol Ryu, who left the laboratory and is at Seoul National University, South Korea. Despite our success to isolate or identify and characterize proper phages and their successful use in rescue of animals in experimental bacterimia, we have indefinitely postponed our own efforts to take the projects to clinical levels. In the past, these trials were being conducted, in consultation with us, by two phage technology companies, Exponential Biotherapies, Inc. and Gangagen, Inc. However, we could not pursue such joint efforts because of more recent NIH guidelines, which discouraged the commercial enterprises to continue interactions with us. Nonetheless, we extended our interest to develop engineered bacteriophages for easy detection of specific bacteria in clinical and environmental samples. We also successfully developed methods to use bacteriophage lambda as a protein display agents. Moreover, we constructed using the lambda-display phage system, a protein 2 hybrid system, called 2lambda system, to study protein-protein interactions <i>in vitro</i>. The phage technology research accomplishments in the last four years are summarized below. (i) Characterization of a lambda phage mutant that survives mammalian circulatory system. In experiments with germ free mice, free from adaptive antibodies to bacteriophage lambda, phage titers in the circulatory system decrease by more than 10(9)pfu within 48 h of intraperitoneal intravenous or oral administration. Based on these observations, we previously used serial passage techniques to select lambda phage mutants, with 13,000-16,000-fold greater capacity to remain in the mouse circulatory system 24h after intraperitoneal injection. These long-circulating phages, had at least three mutations in the original isolates. We have now demonstrated that one of the changes is in the major phage capsid (E) protein, which resulted in the change of glutamic acid to a lysine at residue 158, and is sufficient to confer the long-circulating phenotype that presumably evade the innate immunity. (ii). Characterization of <i>Salmonella</i> and <i>E. coli</i> phages of therapeutic values. We have determined the genome sequences of two lytic bacteriophages, SP6 phi111, which infect <i>Salmonella typhimurium</i> LT2, and phiK1-5, which infects <i>E. coli</i> serotypes K1 and K5. The genome organization of the phages except phi111 is almost identical with the notable exception of the tail fiber genes that confer the different host specificities. It appears that the SP6 and phiK1-5 have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized. The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats. About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage. The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved. (iii) Capsular polysaccharides may be a barrier to phage invasion. <i>E. coli</i> strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cells invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. We have shown that expression of the K1 capsule in <i>E. coli</i> physically blocks infections by T7, a phage that recognizes lipopolysaccharide as the primary receptor.</p><p>2. Highly sensitive detection of bacteria using phage and Quantum dots. With current concerns of antibiotic-resistant bacteria and biodefence, it has become important to rapidly identify infectious bacteria. We developed a rapid and simple method that combines <i>in vivo</i> biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method, provides specific detection of 10 bacterial cells or less per milliliter in experimental and environmental samples, with an approximately 100-fold amplification of the signal over background in one h. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are now feasible since one can label different phages with Quantum dots of different colors.</p><p>3. A lambda-based genetic system to study protein-protein interaction <i>in vitro</i>: 2lambda system. Analyzing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid systems is limited to an <i>in vivo</i> environment. We have developed lambda 2-Hybrid system for studying protein interactions <i>in vitro</i>. Bait and prey are displayed as fusions to the surface protein D of phage lambda that are marked with different selectable drug resistant markers. An interaction of phages <i>in vitro</i> through displayed proteins allows bacterial infection by two phages resulting lysogenic cells, which are detected on double drug resistant bacterial colonies at a very low moi of infection under which coincidental double infection is practically zero. We demonstrated 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 showed the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library.</p