Exploring novel mechanisms of antiviral immunity in bacteria.

项目总结/摘要 对细菌抗病毒防御的研究导致了有价值的工具的发现和实际应用 例如限制酶和CRISPR-Cas。然而，噬菌体防御的多样性 战略是没有得到充分重视和有限的努力已经作出了发现和表征的新的 系统.基于序列的分析表明，有大量的未表征的 噬菌体防御基因尚未被鉴定或实验验证。重要的是， 与抗生素抗性一样，噬菌体抗性可以传播到选择下的人类病原体， 这对噬菌体疗法作为抗生素替代物的实际应用构成了潜在的障碍。因此 更全面地了解噬菌体防御系统的多样性是很重要的， 他们的水平转移潜力。本研究提出了一种高通量的功能选择 方法来鉴定人类微生物宏基因组和菌株集合中的噬菌体防御位点。 Shotgun基因组文库将在大肠杆菌中表达，并选择获得性耐药， 几种类型的E.大肠杆菌噬菌体所提出的策略使用敏感的选择方法， 检测完全或部分抗性，并与深度测序后选择偶联， 吞吐量初步实验表明，小插入（~2 kb）人类宏基因组 文库包含数百个赋予噬菌体T4对E.杆菌在这些数据中， 新的防御系统被发现，如属于保守的单基因系统， 未知函数的家庭，迄今尚未研究。这种基因起源于奈瑟菌， 保护E.大肠杆菌从T4，支持的假设，即人类微生物区系窝藏一个移动的池， 噬菌体抗性这种方法有望立即发现有关一个基本的新信息， 细菌生物学方面。此外，它还将作为确定传播潜力的模式 噬菌体抗性基因的基因。最后，本研究旨在采取遗传和生物化学方法， 详细研究新型防御系统的分子机制，为以下方面提供新的见解： 细菌是如何感知并回应病毒的总之，这项研究将对我们的 对细菌抗病毒免疫的基本认识，并告知噬菌体治疗的进展。 除了研究之外，这项培训计划还将在每周一次的培训中纳入科学交流。 会议，教学和指导机会，以及专业发展。 培训将在马萨诸塞州理工学院进行， 实验室，并将提供成功完成培训的所有方面所需的资源。

Project Summary/Abstract The study of antiviral defenses in bacteria has led to the discovery and practical use of valuable tools such as restriction enzymes and CRISPR-Cas. However, the diversity of bacteriophage defense strategies is not fully appreciated and limited efforts have been made to discover and characterize novel systems. Sequence-based analyses suggest that there is an abundance of uncharacterized bacteriophage-defense genes that have yet to be identified or experimentally validated. Importantly, phage resistance, like antibiotic resistance, may disseminate to human pathogens under selection, posing a potential barrier to the practical use of phage therapy as an antibiotic alternative. Therefore, it is important to have a more complete understanding of the diversity of phage defense systems and their potential to horizontally transfer. This study proposes a high-throughput functional selection approach to identify phage-defense loci in human microbial metagenomes and strain collections. Shotgun genomic libraries will be expressed in Escherichia coli and selected for acquired resistance to several types of E. coli bacteriophage. The proposed strategy uses a sensitive selection method that detects full or partial resistance and is coupled to deep sequencing post-selection to increase throughput. Preliminary experiments have revealed that small-insert (~2 kb) human metagenomic libraries contain hundreds of clones that confer bacteriophage T4 resistance to E. coli. In this data, novel defense systems were discovered such as a single-gene system that belongs to a conserved family of unknown function and has not been studied to date. Originating from Neisseria, this gene protects E. coli from T4, supporting the hypothesis that the human microflora harbors a mobile pool of phage resistance. This method is poised to immediately uncover new information about a fundamental aspect of bacterial biology. In addition, it will serve as a model to determine the dissemination potential of bacteriophage resistance genes. Finally, this study aims to take genetic and biochemical approaches to investigate the molecular mechanisms of novel defense systems in detail, providing new insights into how bacteria sense and respond to their viruses. In all, this study will have long-term impacts on our basic understanding of bacterial antiviral immunity and inform bacteriophage therapy moving forward. In addition to research, this training plan will incorporate scientific communication during weekly meetings and conferences, teaching and mentorship opportunities, and professional development. Training will take place at Massachusetts Institute of Technology in a productive, high-quality research lab, and will provide the resources required for the successful completion of all aspects of the training.