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

项目摘要 霍乱弧菌是影响数百万人的传染性霍乱病的病原体 每年造成约10万人死亡。近年来，越来越明显的是， 水平基因转移事件在非致病性菌株的爆炸性多样化中发挥了至关重要的作用 从中东到现在的致病性埃尔托生物型菌株。毒力和抗生素耐药性 基因通过移动的遗传元件（MGE）在海洋弧菌群落中广泛传播，包括 细菌病毒，质粒和转座子，其中许多永久整合其遗传有效载荷， 基因组此外，霍乱弧菌和病毒之间的动态相互作用直接影响了 霍乱爆发的严重程度，并受到复杂的抗病毒防御系统的有力影响 由MGE传播的系统。因此，病毒捕食和病毒免疫影响霍乱弧菌适应性和致病性， 强调需要更好地了解调节抗病毒防御的水平基因转移过程。 我们的实验室最近发现了一类新的转座因子， CRISPR-Cas系统并通过RNA引导的DNA整合传播，代表了第一个完全的 可编程转座酶这些CRISPR转座子（CRISPR-Tn）系统在弧菌属物种中普遍存在， 迄今为止，我们的研究集中在来自临床霍乱弧菌分离株的代表性转座子上 在2010年海地霍乱疫情期间取样。值得注意的是，在我们最近对基因货物的分析中， 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惊人富集， 这表明这些MGE通过接合质粒水平传播，并通过动员宿主细胞的细胞因子而有益于宿主细胞。 先天免疫系统的丰富补充。我们的中心愿景是确定霍乱弧菌免疫和 致病性受到CRISPR-Tn货物基因的获得的影响，同时还发展RNA引导的 转座酶作为一种新的工具，用于霍乱弧菌的转座酶规模的基因组工程。在目标1中，我们将采用 生物信息学，遗传学和高通量测序，全面研究进化和 CRISPR-Tn系统的机制多样性，并利用最活跃的系统进行高效基因组 在霍乱弧菌中的插入和缺失。在目标2中，我们将分析CRISPR-Tn货物的完整库， 基因，并确定哪些基因簇提供针对弧菌特异性病毒的保护。除了脱落 广泛地了解转座子在弧菌中的功能，这一建议将扩大我们对MGE如何 促进细菌群体内防御系统的快速周转，作为泛免疫系统的一部分。这 鉴于抗生素耐药基因的传播和新的兴趣， 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.