Exploring novel mechanisms of antiviral immunity in bacteria.

项目摘要/摘要 对细菌中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 如限制性内切酶和CRISPR-Cas。然而，噬菌体防御的多样性 策略没有得到充分的理解，发现和刻画小说的努力也有限 系统。基于序列的分析表明，有大量未表征的 噬菌体--尚未被鉴定或实验验证的防御基因。重要的是 噬菌体耐药性，就像抗生素耐药性一样，可能会在选择的情况下传播给人类病原体， 这对噬菌体疗法作为抗生素替代品的实际应用构成了潜在的障碍。因此，它 更全面地了解噬菌体防御系统的多样性和 它们水平转移的潜力。本研究提出了一种高通量的功能选择 在人类微生物元基因组和菌株集合中鉴定噬菌体防御位点的方法。 鸟枪式基因组文库将在大肠杆菌中表达，并对获得的抗性进行筛选 几种类型的大肠杆菌噬菌体。建议的策略使用了一种敏感的选择方法， 检测全部或部分抗性，并与深度测序后选择相结合，以增加 吞吐量。初步实验表明，小插入(~2kb)的人类元基因组 文库包含数百个克隆，这些克隆赋予噬菌体T4对大肠杆菌的抗性。在这些数据中， 新的防御系统被发现，例如属于保守的 一族未知的函数，至今还没有被研究过。起源于奈瑟氏菌的这个基因 保护大肠杆菌免受T4感染，支持这样的假设，即人类微生物群中含有一个可移动的 噬菌体抗性。这种方法有望立即发现关于一个基本问题的新信息 细菌生物学的一个方面。此外，它还将作为确定传播潜力的模型。 抗噬菌体基因。最后，这项研究的目的是采用遗传和生化方法 详细研究新型防御系统的分子机制，为 细菌如何感知和响应它们的病毒。总而言之，这项研究将对我们的 对细菌抗病毒免疫的基本了解，并提示噬菌体治疗的进展。 除了研究之外，这项培训计划还将包括每周的科学交流 会议和会议，教学和指导机会，以及职业发展。 培训将在麻省理工学院进行，进行富有成效的高质量研究 实验室，并将提供成功完成所有方面培训所需的资源。

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.