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

CRISPR相关转座子对霍乱弧菌抗噬菌体免疫的影响

基本信息

批准号：
10556364
负责人：
Samuel Henry Sternberg
金额：
$ 24.3万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10556364
关键词：
Affect Algorithms Antibiotic Resistance Bacteria Bacteriophages Bioinformatics Biological Biological Process CRISPR-associated transposons Candidate Disease Gene Cells Cessation of life Cholera Cholera Toxin Clinical Clustered Regularly Interspaced Short Palindromic Repeats Communities Complement Complex DNA DNA Integration DNA Transposable Elements DNA Transposons Data Set Detection Disease Outbreaks Dysentery Ensure Epidemic Epidemiology Escherichia coli Event Evolution Excision Exhibits Future Gene Cluster Genes Genetic Genetic Recombination Genome Genome engineering Genomics Growth Guide RNA Haiti High-Throughput Nucleotide Sequencing Horizontal Gene Transfer Human Immune system Immunity Individual Infectious Agent Innate Immune System Island Knock-in Knock-out Knowledge Laboratories Life Style Light Link Lytic Microbiology Middle East Mobile Genetic Elements Modification Molecular Monitor Natural Immunity Pathogenicity Pathway interactions Patients Plasmids Play Population Population Dynamics Predatory Behavior Prevalence Process Public Health RNA Research Role Sampling Sequence-Specific DNA Binding Protein Severities Site Specificity System Testing Therapeutic Intervention Transposase Vibrio Vibrio cholerae Vibrio cholerae infection Viral Virulence Virulence Factors Virus Vision Work biotypes cost experimental study fitness gene conservation genetic analysis genetic approach genetic payload genome-wide immunological diversity improved insertion/deletion mutation interest marine microbial microorganism novel novel strategies nuclease pathogen prevent reconstitution resistance gene tool transmission process

项目摘要

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.

项目概要霍乱弧菌是传染性腹泻病霍乱的病原体，影响数百万人每年造成约 100,000 人死亡。近年来人们越来越清楚地认识到水平基因转移事件在非致病菌株的爆炸性多样化中发挥了至关重要的作用从中东引入当今的致病性 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（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，其中许多将其遗传负载永久整合到基因组。此外，霍乱弧菌和病毒之间的动态相互作用直接影响持续时间和霍乱爆发的严重程度，并受到复杂的抗病毒防御系统的强烈影响由 MGE 传播的系统。因此，病毒捕食和病毒免疫影响霍乱弧菌的适应性和致病性，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元件，可编码核酸酶缺陷 CRISPR-Cas 系统并通过 RNA 引导的 DNA 整合进行传播，代表了第一个完全可编程转座酶。这些 CRISPR 转座子 (CRISPR-Tn) 系统在弧菌物种中普遍存在，迄今为止，我们的研究主要集中在源自临床霍乱弧菌分离株的代表性转座子 2010 年海地霍乱流行期间采样。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，我们发现抗病毒防御基因显着富集，表明这些 MGE 通过接合质粒水平传播，并通过动员丰富的先天免疫系统补充。我们的中心愿景是确定霍乱弧菌免疫和致病性受到 CRISPR-Tn 货物基因获取的影响，同时还开发了 RNA 引导的转座酶作为霍乱弧菌千碱基级基因组工程的新工具。在目标 1 中，我们将雇用生物信息学、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实现高效基因组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可以针对弧菌特异性病毒提供保护。超越脱落广泛地阐述转座子在弧菌中的功能，该提案将扩大我们对 MGE 的理解作为泛免疫系统的一部分，促进细菌群体内防御系统的快速更新。这鉴于抗生素抗性基因的传播和新的兴趣，这一主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。