Impact of CRISPR-associated transposons on anti-phage immunity in Vibrio cholerae

项目总结 霍乱弧菌是影响数百万人的传染性腹泻疾病霍乱的病原体。 个人和死因每年约100,000人死亡。近年来，越来越明显的是， 水平基因转移事件在非致病毒株的爆炸性多样化中发挥了关键作用 从中东进入今天的致病性El Tor生物型菌株。毒力和抗生素耐药性 基因通过移动遗传元件(MGES)在海洋弧菌群落中广泛传播，包括 细菌病毒、质粒和转座子，其中许多永久地将它们的基因有效载荷整合到 基因组。此外，霍乱弧菌与病毒之间的动态相互作用直接影响持续时间 和霍乱暴发的严重性，并受到复杂的抗病毒防御体系的影响 由MGES传播的系统。因此，病毒捕食和病毒免疫影响霍乱弧菌的适合性和致病性， 突显了更好地了解调节抗病毒防御的水平基因转移过程的必要性。 我们实验室最近发现了一类新的编码缺乏核酸酶的转座元件 CRISPR-CAS系统并通过RNA引导的DNA整合传播，这是第一个完整的 可编程转座酶。这些CRISPR-转座子(CRISPR-TN)系统普遍存在于弧菌物种中， 到目前为止，我们的研究主要集中在来自霍乱弧菌临床分离株的一个具有代表性的转座子上。 在2010年海地霍乱疫情期间采集的样本。值得注意的是，在我们最近对遗传货物的分析中 在一组更大的CRISPR转座子中发现，我们发现了抗病毒防御基因的显著丰富， 这表明这些MGES通过结合质粒水平传播，并通过动员一种 丰富的先天免疫系统补体。我们的中心目标是确定霍乱弧菌的免疫力和 致病性受到CRISPR-TN货物基因的获取的影响，同时也是由RNA引导的 转座酶作为霍乱弧菌千碱基规模基因组工程的新工具。在目标1中，我们将使用 生物信息学、遗传学和高通量测序，以全面研究进化和 CRISPR-TN系统的机械多样性，并利用最活跃的系统进行高效基因组 霍乱弧菌的插入和缺失。在目标2中，我们将分析CRISPR-TN货物的完整曲目 基因，并确定哪些基因簇提供针对弧菌特定病毒的保护。超越脱落 广泛地阐明弧菌中转座子的功能，这一建议将扩大我们对MGES如何 促进细菌种群内防御系统的快速更新，作为泛免疫系统的一部分。这 鉴于抗生素耐药性基因的传播和重新引起的兴趣，这一话题变得越来越重要 用于霍乱弧菌和许多其他病原微生物的噬菌体治疗。

PROJECT SUMMARY Vibrio cholerae is the causative agent of the infectious diarrheal disease, cholera, which affects several million individuals and causes ~100,000 deaths, annually. It has become increasingly clear in recent years that horizontal gene transfer events played a crucial role in the explosive diversification of a non-pathogenic strain from the Middle East into the present-day pathogenic El Tor biotype strain. Virulence and antibiotic resistance genes are broadly disseminated within marine Vibrio communities by mobile genetic elements (MGEs), including bacterial viruses, plasmids, and transposons, many of which permanently integrate their genetic payloads into the genome. Furthermore, dynamic interactions between V. cholerae and viruses directly impact the duration and severity of cholera outbreaks, and are potently influenced by the complex repertoire of antiviral defense systems spread by MGEs. Thus, viral predation and viral immunity affect V. cholerae fitness and pathogenicity, highlighting the need to better understand horizontal gene transfer processes that modulate antiviral defense. Our laboratory recently discovered a new class of transposable elements that encode nuclease-deficient CRISPR-Cas systems and spread via RNA-guided DNA integration, representing the first example of a fully programmable transposase. These CRISPR-transposon (CRISPR-Tn) systems are prevalent in Vibrio species, and our studies have thus far focused on a representative transposon derived from a clinical V. cholerae isolate sampled during the 2010 Haiti cholera epidemic. Remarkably, during our recent analyses of the genetic cargos found within a larger set of CRISPR-transposons, we uncovered a striking enrichment in antiviral defense genes, suggesting that these MGEs spread horizontally via conjugative plasmids and benefit host cells by mobilizing a rich complement of innate immune systems. Our central vision is to determine how V. cholerae immunity and pathogenicity is influenced by the acquisition of CRISPR-Tn cargo genes, while also developing RNA-guided transposases as a new tool for kilobase-scale genome engineering in V. cholerae. In Aim 1, we will employ bioinformatics, genetics, and high-throughput sequencing to comprehensively investigate the evolutionary and mechanistic diversity of CRISPR-Tn systems, and leverage the most active systems for high-efficiency genomic insertions and deletions in V. cholerae. In Aim 2, we will analyze the complete repertoire of CRISPR-Tn cargo genes and determine which gene clusters provide protection against Vibrio-specific viruses. Beyond shedding light broadly on the function of transposons in Vibrio, this proposal will expand our understanding of how MGEs promote rapid turnover of defense systems within bacterial populations as part of the pan-immune system. This topic is of increasingly critical importance, given the spread of antibiotic resistance genes and renewed interest in phage therapy for V. cholerae and numerous other pathogenic microorganisms.