Mining the phage playbook to create a potent, generic phage therapy

项目摘要 噬菌体治疗，用细菌靶向病毒治疗细菌感染的实践，噬菌体（或 噬菌体）是一种有前途的和迫切需要的抗生素替代品。保持这种方法的主要挑战是 从广泛采用的情况来看，噬菌体治疗需要针对每个国家的感染菌株进行定制， 耐心，一个缓慢且劳动密集型的过程。这一要求是由于宿主范围非常狭窄 即使是在关系密切的菌株中也是如此。驱动噬菌体宿主的主要因素 范围是不均匀分布的细菌抗噬菌体免疫机制的巨大集合 在细菌菌株之间。然而，细菌和噬菌体之间潜在的分子军备竞赛， 产生了一组同样令人印象深刻的相应噬菌体反防御途径， 噬菌体已经进化出克服大多数细菌防御的机制。类似于他们 细菌对应物，每个噬菌体菌株仅编码现有反防御的极小部分，因此， 这解释了个体寄生范围的狭窄。开发一种噬菌体治疗方法， 将天然存在的噬菌体溶液转化为“超级噬菌体混合物”将能够生产现成的 噬菌体处理具有极大扩展的物种范围和阻止细菌抗性的能力。 在这里，我建议开发一个利用现有噬菌体反防御机制的管道， 这是一种针对机会致病菌铜绿假单胞菌的强大的原理验证噬菌体混合物。实现 为了实现这一愿景，我将采用实验性基因组学方法来绘制临床相关P. 分离铜绿假单胞菌，从而确定噬菌体在感染期间将遇到哪些细菌防御。 然后我将开发一个强大的，高通量的筛选，以确定现有的噬菌体反防御机制 能够克服这些细菌防御的细菌。最后，我将创建一个超级噬菌体鸡尾酒编码广泛的 收集具有感染广泛的铜绿假单胞菌菌株的能力的反防御基因盒。 这些研究试图利用细菌噬菌体分子军备竞赛的现有生物学基础， 克服了噬菌体疗法发展中的一个主要障碍。这项工作将提供前所未有的洞察力 深入了解细菌免疫和噬菌体反防御的广度和多样性， 新的生物学机制将在未来的研究中得到表征。工程化噬菌体混合物还 构成了一个创新的实验系统，可用于回答有关病毒的基本问题， 种群多样性与进化这项初步研究将作为噬菌体的发展蓝图 治疗其他多重耐药的机会性病原体。

PROJECT SUMMARY Phage therapy, the practice of treating bacterial infections with bacteria-targeting viruses, bacteriophage (or phage), is a promising and urgently needed alternative to antibiotics. A major challenge holding this approach back from widespread adoption is that phage treatments need to be customized for the infecting strains in each patient, a slow and labor-intensive process. This requirement arises from the exquisitely narrow host-range that many phages display, even among closely related bacterial strains. A major factor driving phage host range is the immense collection of bacterial anti-phage immune mechanisms that are unevenly distributed across bacterial strains. However, the underlying molecular arms race between bacteria and phage has given rise to an equally impressive set of corresponding phage counter-defense pathways, and thus collectively phage have already evolved mechanisms by which to overcome most bacterial defenses. Similar to their bacterial counterparts, each phage strain encodes only a miniscule fraction of existing counter-defenses, thus explaining the narrow host-range of individual phages. Developing a phage treatment that could amass these naturally occurring phage solutions into a “super phage cocktail” would enable production of an off-the-shelf phage treatment with a greatly expanded species range and the ability to forestall bacterial resistance. Here, I propose developing a pipeline leveraging existing phage counter-defense mechanisms to create a powerful proof-of-principle phage cocktail for the opportunistic pathogen, Pseudomonas aeruginosa. To realize this vision, I will take an experimental genomic approach to map the immune system of clinically relevant P. aeruginosa isolates and thereby determine which bacterial defenses the phage will encounter during infections. I will then develop a powerful, high throughput screen to identify existing phage counter-defense mechanisms that can overcome these bacterial defenses. Finally, I will create a super phage cocktail encoding an extensive collection of counter-defense gene cassettes with the ability to infect a broad set of P. aeruginosa strains. These studies seek to leverage the existing biology underlying the bacterial-phage molecular arms race to overcome a major hurdle in the development of phage therapy. This work will provide unprecedented insight into the breadth and diversity of both bacterial immunity and phage counter-defenses and uncover a multitude of novel biological mechanisms to be characterized in future studies. The engineered phage cocktail also constitutes an innovative experimental system that can be used to answer fundamental questions about viral population diversity and evolution. This initial study will serve as the blue print for development of phage therapy for other multi-drug resistant opportunistic pathogens.