Determining the mechanism of heritable inactivation of bacterial immunity

确定细菌免疫可遗传失活的机制

• 批准号: 10216179
• 负责人: Senen Dario Mendoza
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2021-07-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216179
• 关键词: Antibiotics; Autoimmunity; Bacteria; Bacteriophage Genetics; Bacteriophages; Biological; Biology; CRISPR interference; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communities; Coupled; Cultural Diversity; DNA; DNA Modification Process; DNA Restriction Enzymes; DNA Restriction-Modification Enzymes; Development; Drug Targeting; Educational Status; Endonuclease I; Ensure; Environment; Epigenetic Process; Exhibits; Generations; Genes; Genetic; Genetic Transcription; Genome; Gentamicins; Goals; Growth; Guide RNA; Half-Life; Head; Heat-Shock Response; Heritability; Hour; Immune; Immune system; Immunity; Immunology; Infection; Inherited; Knock-out; Laboratories; Leadership; Libraries; Literature; Lytic; Maintenance; Mass Spectrum Analysis; Measures; Mediating; Memory; Mentors; Mentorship; Messenger RNA; Methods; Microbiology; Microscopy; Modification; Molecular; Molecular Profiling; Mutation; Natural Immunity; Nature; Operon; Organism; Other Genetics; Outcome; Output; Parasites; Peptide Hydrolases; Phase; Phenotype; Positioning Attribute; Post-Translational Protein Processing; Postdoctoral Fellow; Prions; Process; Protein Subunits; Proteins; Pseudomonas aeruginosa; Publications; Regulation; Reporting; Research; Scheme; Science; Scientist; Source; Stress; Supervision; Temperature; Testing; Therapeutic; Time; Training; Transcript; Translations; Virus; Western Blotting; Work; causal variant; cold temperature; drug resistant pathogen; epigenetic regulation; experimental study; fitness; gene repression; genome sequencing; genome-wide; graduate student; inhibitor/antagonist; medical schools; member; next generation; novel; novel strategies; nuclease; polysome profiling; prevent; programs; response; skills; sup35; trait; transcriptome sequencing; undergraduate student; whole genome

Abstract Bacterial immune systems represent a potent barrier to genetic parasites including viruses (bacteriophages, or phages). Harboring immunity against these often-lethal invaders represents a clear fitness benefit. Unexpectedly, during growth at elevated temperatures (i.e. 42 °C), many Pseudomonas aeruginosa strains inactivate a potent anti-bacteriophage immune system, type I restriction-modification (R-M). Remarkably, the inactivation of restriction endonuclease (iREN) phenotype is heritable and persists for at least 60 generations after return to a low temperature (i.e. 37 or 30 °C), well after the environmental stress has ended. This phenomenon is an uncharacterized example of epigenetic regulation and cellular memory in bacteria. Understanding iREN's mechanism could reveal novel bacterial biology, drug targets, and approaches to potentiate bacteriophage therapeutics. This proposal's objective is to understand the molecular basis of iREN and identify the genes involved in R-M inactivation and memory. This will be accomplished through 1) the use of fluorescently tagged R-M proteins to determine their fate during iREN, and 2) the use of RNA sequencing and a CRISPRi transcriptional repression screen coupled with a phage selection, to collectively observe and perturb operons involved in iREN. These aims are essential to understanding how bacteria mount this regulatory response and how this response persists for 60 generations. Our findings may detail a novel cellular strategy for regulation and epigenetic inheritance, as well as explain how inactivation of immunity could be evolutionarily beneficial to bacteria. This project will be conducted in the Bondy-Denomy lab in the Department of Microbiology and Immunology at UCSF's School of Medicine. The Bondy-Denomy lab is a multicultural and intellectually diverse laboratory where undergraduates, graduate students, and postdoctoral fellows work in approximately a 1:1:1 ratio. This distribution of training levels ensures that graduate students enjoy the opportunity to both find mentorship from various perspectives, as well as develop the leadership skills needed to become a PI by mentoring others. I have been a member of the Bondy-Denomy lab for 2.5 years, during which I have spearheaded a collaborative project under Dr. Bondy-Denomy's supervision, culminating in a first author publication. With two senior graduate students graduating shortly, I am rising to a position of leadership in the lab. The culture of UCSF is guided by our PRIDE values: professionalism, respect, integrity, diversity, and excellence. These values serve as both an ideal and the reality of our nurturing community encouraging an environment of collaboration, mentorship, and rigorous standards for science. By training in this environment, my scientific and leadership skills will be cultivated to become an excellent and responsible scientist at the head of an academic research group.

抽象的 细菌免疫系统代表了遗传寄生虫的潜在障碍，包括病毒（噬菌体或 噬菌体）。对这些经常致命的入侵者的免疫力具有明显的健身益处。 出乎意料的是，在升高温度（即42°C）的生长期间，许多铜绿假单胞菌菌株 灭活潜在的抗细菌免疫系统，I型限制性修饰（R-M）。值得注意的是 限制性核酸内切酶（IREN）表型的失活是可遗传的，至少有60代 返回低温（即37或30°C）后，在环境应力结束后。这 现象是细菌中表观遗传调节和细胞记忆的一个未表征的例子。 了解Iren的机制可以揭示新的细菌生物学，药物靶标和方法 增强噬菌体疗法。该提议的目标是了解Iren的分子基础 并确定与R-M灭活和记忆有关的基因。这将通过1）使用 荧光标记的R-M蛋白在IREN期间确定其命运，2）使用RNA测序和A CRISPRI转录表达式屏幕加上噬菌体选择，共同观察和扰动 参与Iren的操纵子。这些目的对于了解细菌如何进行这种调节至关重要 响应以及这种响应如何持续60代。我们的发现可能会详细介绍一种新颖的蜂窝策略 调节和表观遗传遗传，以及解释如何在进化上灭活免疫力 对细菌有益。 该项目将在微生物学系的债券 - 降解实验室中进行 UCSF医学院的免疫学。债券 - 脱瘤实验室是多元文化和智力上的潜水 本科生，研究生和博士后研究员在大约1：1：1的实验室工作 比率。培训水平的分配确保研究生享受俩找到的机会 从各种角度来衡量，以及发展成为PI所需的领导能力 指导别人。我一直是Bondy-DeNomy Lab的成员已有2。5年了，在此期间 带头在邦迪·迪尼科（Bondy-Denomy）的监督下率领一个合作项目，最终在第一作者中 发布。我很快就会有两名高级毕业生毕业，我正在担任领导地位 实验室。 UCSF的文化以我们的骄傲价值观为指导：专业，尊重，正直，多样性和 卓越。这些价值观既是理想，又是我们培育社区的现实，鼓励一个 协作，心态和严格的科学标准的环境。通过在这种环境中的培训， 我的科学和领导能力将被培养为成为一名出色而负责任的科学家 一个学术研究小组。