Exploring novel mechanisms of antiviral immunity in bacteria.

探索细菌抗病毒免疫的新机制。

• 批准号: 10208818
• 负责人: Christopher Vassallo
• 金额: $ 6.64万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2022-10-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10208818
• 关键词: Address; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Antiviral Agents; Bacteria; Bacterial Genome; Bacteriophage T4; Bacteriophages; Base Sequence; Biochemical; Bioinformatics; Biological; Biology; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communication; Complex; Coupled; DNA Restriction-Modification Enzymes; Data; Defense Mechanisms; Development; Ecosystem; Educational process of instructing; Escherichia coli; Family; Future; Genes; Genetic; Genomic Library; Horizontal Gene Transfer; Human; Human Microbiome; Immune; Immune system; Immunity; Infection; Institutes; Knowledge; Lead; Libraries; Massachusetts; Mentorship; Metagenomics; Methods; Microbial Genetics; Modeling; Molecular; Molecular Biology; Neisseria; Nucleic Acids; Organism; Race; Research; Research Training; Resistance; Resources; Shotguns; Signal Transduction; System; Technology; Training; Virus; Virus Diseases; antibiotic resistant infections; antiviral immunity; arm; bacterial genetics; base; deep sequencing; experimental study; fighting; genomic locus; human microbiota; human pathogen; insight; meetings; metagenome; microbial; novel; pathogen; pathogenic bacteria; resistance gene; response; restriction enzyme; symposium; tool; tool development

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.

项目概要/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使用 例如限制性内切酶和 CRISPR-Cas。然而，噬菌体防御的多样性 策略尚未得到充分认识，并且在发现和表征新颖性方面所做的努力有限 系统。基于序列的分析表明存在大量未表征的 尚未鉴定或实验验证的噬菌体防御基因。重要的是， 噬菌体抗性，就像抗生素抗性一样，可能会在选择下传播给人类病原体， 对噬菌体疗法作为抗生素替代品的实际应用构成了潜在障碍。因此，它 对噬菌体防御系统的多样性有更全面的了解非常重要 他们横向转移的潜力。本研究提出了一种高通量功能选择 识别人类微生物宏基因组和菌株集合中噬菌体防御位点的方法。 鸟枪法基因组文库将在大肠杆菌中表达，并根据获得性耐药性进行选择 几种类型的大肠杆菌噬菌体。所提出的策略使用敏感选择方法 检测全部或部分耐药性，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（~2 kb）人类宏基因组 文库包含数百个赋予噬菌体 T4 大肠杆菌抗性的克隆。在这份数据中， 发现了新的防御系统，例如属于保守的单基因系统 家族的功能未知，迄今为止尚未被研究过。该基因源自奈瑟氏球菌 保护大肠杆菌免受 T4 侵害，支持人类微生物群落拥有一个可移动的细菌池的假设 噬菌体抗性。这种方法有望立即发现有关基本面的新信息 细菌生物学的一个方面。此外，它将作为确定传播潜力的模型 噬菌体抗性基因。最后，本研究旨在采用遗传和生化方法 详细研究新型防御系统的分子机制，提供新的见解 细菌如何感知病毒并对其做出反应。总而言之，这项研究将对我们产生长期影响。 对细菌抗病毒免疫的基本了解，并为噬菌体治疗的发展提供信息。 除了研究之外，该培训计划还将在每周期间纳入科学交流 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，并将提供成功完成培训各方面所需的资源。