Investigating the mechanisms that make jumbophages impervious to bacterial immune systems

研究使巨噬细胞不受细菌免疫系统影响的机制

• 批准号: 10663359
• 负责人: Joseph Bondy-Denomy
• 金额: $ 45.83万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-11 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10663359
• 关键词: Alleles; Antibiotics; Back; Bacteria; Bacteriophages; Biological; Biology; Biotechnology; Cations; Cell Nucleus; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cryo-electron tomography; DNA; DNA Restriction-Modification Enzymes; DNA biosynthesis; Detection; Engineering; Enzymes; Exclusion; Experimental Genetics; Family; Future; Genes; Genetic Screening; Genetic Transcription; Genome; Goals; Immune; Immune Evasion; Immune Targeting; Immune system; Immunity; In Vitro; Infection; Ion Channel; Lytic; Medicine; Microbe; Microscopy; Modeling; Modification; Molecular; Molecular Target; Mutation; Nuclear Pore; Organelles; Pathway interactions; Planets; Production; Property; Protein Import; Proteins; Proteomics; Pseudomonas aeruginosa; Role; Structural Models; Structure; Superbug; System; Testing; Therapeutic; Virus; Work; drug resistant pathogen; experimental study; fascinate; fighting; genetic selection; human pathogen; inhibitor; innovation; member; mutant; next generation; novel; nuclease; pathogen; prevent; programs; protein purification; protein transport; response; restriction enzyme; scaffold; success

Project Summary The viruses that infect bacteria, called bacteriophages (phages), are robust killers. In response to the frequent threat of phage infection, bacteria have developed a suite of anti-phage immune mechanisms, such as restriction-modification and CRISPR-Cas enzymes. Phages have emerged as promising alternatives to antibiotics in our current “superbug” crisis, but immune systems are a barrier for successful phage replication. Broad-spectrum phages that evade immune detection and kill multiple isolates of antibiotic-resistant pathogens may prove essential in this fight. We screened 12 obligately lytic phages infecting the prominent antibiotic- resistant pathogen Pseudomonas aeruginosa to identify phages with the ability to broadly evade DNA-targeting immune systems CRISPR-Cas and restriction enzymes. Jumbophage ΦKZ evaded all six DNA-targeting systems tested, making it the strongest “anti-immune” phage identified to date. The mechanisms behind pan- immune evasion for this phage family will be investigated here, with the goal of making fundamental discoveries at the phage host-interface that could benefit phage therapies and other biotechnologies in the future. ΦKZ is a jumbophage with a 280 kb genome, has many relatives that infect other Gram negative pathogens, and is outstanding in its ability to evade bacterial nucleolytic immune systems. Immune evasion is enabled by the assembly of a phage-encoded proteinaceous nucleus-like shell (“phage nucleus”) that serves as a replicative compartment. However, it is unknown how this phage protects its genome prior to the phage nucleus being assembled and subsequently, how protein inclusion/exclusion is regulated. We have identified phage proteins that are ejected with the genome and hypothesize that an “injected structure” (IS) creates a DNA- containing organelle that occludes immune nucleases. Understanding how ejected proteins can rapidly shield DNA from numerous host nucleases, and how the host fights back against the IS with novel immune systems likely represents fundamentally new phage-host interaction paradigms. Next, the nascent phage nucleus assembles adjacent to the IS and receives the phage genome. Subsequently, the phage nucleus imports proteins involved in DNA replication and transcription, while excluding immune nucleases, through unknown mechanisms. A genetic screen in our lab has identified the first phage mutants defective in protein import, with mutations in a single gene. Structure predictions suggest that the encoded protein may be homologous to the conserved TRPV family of ion channels. We will determine its subcellular localization, interaction partners and in vitro properties with the goal of elucidating how a “nuclear pore” could work in a phage. In sum, our work here will unveil new phage biology driven by the co-evolution of host and virus, leading to innovative and potentially transferrable mechanisms for enhancing phage success in the fight against deadly pathogens.

项目摘要 感染细菌的病毒被称为噬菌体（噬菌体），是强大的杀手。响应频率 噬菌体感染的威胁，细菌已经开发了一套抗流量免疫机制，例如 限制性修饰和CRISPR-CAS酶。噬菌体已经成为承诺的替代方案 在我们目前的“超级细菌”危机中，抗生素是成功复制噬菌体的障碍。 远程逃避免疫检测并杀死多种抗生素耐药性病原体的宽光谱噬菌体 可能在这场战斗中被证明是必不可少的。我们筛选了12种裂解的裂纹噬菌体，感染了突出的抗生素 - 抗性病原体假单胞菌铜绿可识别具有广泛逃避DNA靶向的能力的噬菌体 免疫系统CRISPR-CAS和限制性酶。 Jumbophageφkz逃避了所有六个DNA靶向 经过测试的系统，使其成为迄今为止确定的强烈“反免疫”噬菌体。 Pan-背后的机制 这个噬菌体家族的免疫进化将在这里进行调查，目的是进行基本发现 在噬菌体宿主界面上，将来可能受益于噬菌体疗法和其他生物技术。 φkz是具有280 kb基因组的巨型噬菌体，有许多感染其他革兰氏阴性的亲戚 病原体，它在逃避细菌核溶解免疫系统的能力方面非常出色。免疫进化是 通过组装噬菌体编码的蛋白质核的壳（“噬菌体核”）的组装来启用 复制室。但是，尚不清楚该噬菌体在噬菌体核之前如何保护其基因组 被组装，随后，如何调节蛋白质包含/排除。我们已经确定了噬菌体 被基因组喷射并假设“注射结构”（IS）产生DNA-的蛋白质 包含遮挡免疫核酸的细胞器。了解弹出蛋白如何迅速屏蔽 来自众多宿主核的DNA，以及宿主如何通过新颖的免疫与IS进行反击 系统可能代表了新的噬菌体 - 主宿主相互作用范例。接下来，新生的噬菌体 核聚集到与IS相邻并接收噬菌体基因组。随后，噬菌体核进口 通过未知 机制。我们实验室中的遗传筛查确定了蛋白质进口中的第一个噬菌体突变体， 与单个基因中的突变。结构预测表明，编码的蛋白质可能与 离子通道的配置TRPV家族。我们将确定其亚细胞定位，相互作用伙伴 并旨在阐明“核孔”如何在噬菌体中起作用。总之，我们的工作 这里将揭示由宿主和病毒共同发展驱动的新的噬菌体生物学，从而导致创新和 潜在地转移了在对抗致命病原体的战斗中增强噬菌体成功的机制。