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: 10503219
• 负责人: Joseph Bondy-Denomy
• 金额: $ 48.1万
• 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/10503219
• 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; Evolution; Exclusion; Family; Future; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Goals; Hand; 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; Sum; Superbug; System; Testing; Therapeutic; Virus; Work; drug resistant pathogen; experimental study; fascinate; fight against; fighting; genetic selection; human pathogen; inhibitor; innovation; member; mutant; next generation; novel; nuclease; pathogen; prevent; programs; 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，是强大的杀手。为了应对频繁的 面对噬菌体感染的威胁，细菌已经发展出一套抗噬菌体免疫机制，例如 限制性内切酶修饰和CRISPR-Cas酶。噬菌体已经成为有希望的替代品 抗生素在我们目前的“超级细菌”危机中，但免疫系统是成功复制噬菌体的障碍。 逃避免疫检测并杀死多个抗药性病原体菌株的广谱噬菌体 在这场战斗中可能被证明是至关重要的。我们筛选了12个感染主要抗生素的专性裂解噬菌体- 抗病原菌铜绿假单胞菌鉴定具有广泛逃避DNA靶向能力的噬菌体 免疫系统CRISPR-CA和限制性内切酶。巨型噬菌体ΦKZ躲过了所有六个dna目标 系统测试，使其成为迄今发现的最强的“抗免疫”噬菌体。泛欧经济一体化背后的机制 这里将研究这个噬菌体家族的免疫逃逸，目的是做出根本性的发现。 在噬菌体宿主界面，这可能会在未来受益于噬菌体疗法和其他生物技术。 ΦKZ是一种具有280kb基因组的巨噬菌体，有许多亲戚感染其他革兰氏阴性菌 病原体，突出的是它有能力躲避细菌的核溶解免疫系统。免疫逃避是 由一种噬菌体编码的蛋白核样外壳(“噬菌体核”)的组装实现，该噬菌体核充当 一个复制的隔间。然而，这种噬菌体是如何在噬菌体核之前保护其基因组的，目前尚不清楚。 组装后，蛋白质包含/排除如何被调控。我们已经鉴定出噬菌体 与基因组一起喷射出来的蛋白质，并假设“注入结构”(IS)创造了一种DNA- 含有封闭免疫核酸酶的细胞器。了解喷射出的蛋白质如何快速屏蔽 来自多种宿主核酸酶的DNA，以及宿主如何用新的免疫力反击IS 这些系统很可能代表了全新的噬菌体-宿主相互作用范例。接下来，新生的噬菌体 细胞核在IS附近组装，并接收噬菌体基因组。随后，噬菌体核进口 参与DNA复制和转录的蛋白质，但不包括免疫核酸酶，通过未知的 机制。我们实验室的基因筛查已经确定了第一个在蛋白质输入方面有缺陷的噬菌体突变体， 与单个基因的突变有关。结构预测表明，编码的蛋白质可能与 保守的TRPV离子通道家族。我们将确定它的亚细胞定位、交互伙伴 以及体外性质，目的是阐明“核孔”如何在噬菌体中工作。总而言之，我们的工作 在这里将揭开由宿主和病毒共同进化驱动的新的噬菌体生物学，导致创新和 潜在的可转移机制，用于提高噬菌体在对抗致命病原体方面的成功。