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.

摘要 细菌免疫系统代表了对包括病毒（噬菌体或噬菌体）在内的遗传寄生虫的有效屏障。 ）。对这些致命的入侵者拥有免疫力是一个明显的健康益处。 出乎意料的是，在高温（即42 °C）下生长期间，许多铜绿假单胞菌菌株 一种有效的抗噬菌体免疫系统，I型限制修饰（R-M）。值得注意的是， 限制性内切酶（iREN）表型失活是可遗传的，并持续至少60代 在环境胁迫结束后很久，恢复到低温（即37或30 °C）后。这 这种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，药物靶点和方法， 加强噬菌体治疗。该提案的目的是了解iREN的分子基础 并确定参与R-M失活和记忆的基因。这将通过以下方式实现：1）使用 荧光标记的R-M蛋白，以确定它们在iREN期间的命运，以及2）使用RNA测序和 CRISPRi转录抑制筛选结合噬菌体选择，共同观察和干扰 参与iREN的操纵子。这些目标对于理解细菌如何建立这种调节机制至关重要。 以及这种反应如何持续60代。我们的发现可能详细说明了一种新的细胞策略， 调节和表观遗传，以及解释免疫失活如何在进化上 对细菌有益。 该项目将在微生物学系的Bondy-Denomy实验室进行， 加州大学旧金山分校医学院的免疫学。Bondy-Denomy实验室是一个多元文化和知识多样的 实验室，本科生，研究生和博士后研究员在大约1：1：1的工作 比例这种培训水平的分布确保了研究生有机会既找到 从不同的角度指导，以及发展成为PI所需的领导技能， 指导他人。我已经成为邦迪-Denomy实验室的一员2.5年了，在此期间，我 在Bondy-Denomy博士的监督下，率先开展了一个合作项目，最终产生了第一作者 出版物随着两名高年级研究生即将毕业，我正在上升到一个领导职位， 实验室UCSF的文化是由我们的骄傲价值观指导：专业，尊重，诚信，多样性， 卓越这些价值观既是理想，也是我们培育社区的现实，鼓励 协作环境，指导和严格的科学标准。通过在这种环境中训练， 我的科学和领导能力将被培养成为一个优秀的和负责任的科学家在头部 一个学术研究小组的成员。