Elucidation of the mechanisms of jumbophage genome protection during infection

面对对大多数现有抗生素具有耐药性的病原菌的公共卫生危机， （细菌病毒;细菌），自然感染和杀死细菌，代表了一个有希望的替代品，用作 抗菌剂然而，实现这一转化目标的一个重大障碍是过多的 细菌抵抗噬菌体感染的免疫机制。要克服这一障碍， 具有强大的抗免疫能力，可杀死多种耐药病原菌。 在这方面，Φ KZ样巨噬细胞（基因组> 200 kb的噬菌体）作为一个家族出现， 在整个感染过程中阻碍多种细菌溶核免疫系统的能力， 感染最重要的革兰氏阴性病原体的成员。巨噬细胞ΦKZ是一个广泛的宿主范围 多重耐药病原菌铜绿假单胞菌的杀手及主导模式 巨噬细胞家族的噬菌体免疫逃避主要是通过组装 噬菌体编码的蛋白质核样“壳“隔室，其封装并屏蔽复制的 噬菌体基因组然而，目前尚不清楚这种噬菌体如何在壳组装之前保护其基因组。 这项提议的长期目标是了解用于保护被排出的基因组的机制， 类似Jumbophages-like的巨噬细胞-初步证据（来自我们和其他人）表明， 在感染细胞中的类似位置处的区室作为弹出的巨噬细胞基因组（在壳组装之前）。 目前，这些复合物的组成和生物学意义仍然未知。我假设 该复合物（在此称为“弹出结构”）产生包含DNA的细胞器， 保护排出的噬菌体基因组免受免疫核酸酶的侵害。我将首先使用荧光显微镜， 冷冻电子断层扫描，以便在壳体组装之前观察感染状态。从这些研究中，我将获得 高分辨率的结构洞察这些隔间的生物起源和组织。与此同时，我将 使用质谱法来揭示这些组件的成分，并随后询问 这些成分在支持噬菌体感染中的功能作用。从我的多学科研究来看， 从根本上揭示新的噬菌体生物学，以及创新和潜在的可转移机制， 以增强噬菌体在对抗病原菌中的成功率。 我将在加州大学旧金山分校进行这项工作，该研究所拥有最先进的研究设施，并培养了一个 学术严谨和协作的研究环境。契约经济中的共同监督 Agard实验室将确保在噬菌体生物学，遗传学和生物物理学的全面培训。通过我的课程， 研究，我将接受广泛的培训，随后获得微生物学不同领域的专业知识， 分子生物学，生物物理学和结构生物学，我需要实现我的博士后培养目标， 随后开创了一项独立研究计划。

In the face of a public health crisis of pathogenic bacteria resistant to most existing antibiotics, bacteriophages (bacterial viruses; phages) that naturally infect and kill bacteria, represent a promising alternative for use as antimicrobials. However, a significant obstacle to realizing this translational goal is presented by the plethora of immune mechanisms in bacteria to resist phage infections. Overcoming this obstacle necessitates phages equipped with robust anti-immune capabilities to kill multiple isolates of antibiotic-resistant pathogenic bacteria. In that regard ΦKZ-like jumbophages (phages with genomes > 200kb) emerge as a family with an outstanding ability to thwart a multitude of bacterial nucleolytic immune systems throughout infection and numerous family members that infect most important Gram negative pathogens. The jumbophage ΦKZ is a broad-host range killer of the multi-antibiotic resistant pathogenic bacteria Pseudomonas aeruginosa and the leading model phage for the jumbophage family. Immune evasion is achieved largely through the assembly of a phage-encoded proteinaceous nucleus-like “shell “compartment that encapsulates and shields the replicating phage genome. However, it is unknown how this phage protects its genome prior to the assembly of the shell. The long term objective of this proposal is to understand the mechanisms used to guard the ejected genome of ФKZ-like jumbophages–preliminary evidence (from us and others) suggests the assembly of a macromolecular compartment in infected cells at similar locations as the ejected jumbophage genome (prior to shell assembly). Currently, the composition and biological significance of these complexes remain unknown. I hypothesize that this complex (termed herein the ‘ejected structure’) creates a DNA-containing organelle that encapsulates and protects the ejected phage genome from immune nucleases. I will first use fluorescence microscopy and cryo-electron tomography to visualize the infection state prior to shell assembly. From these studies I will gain high resolution structural insight into the biogenesis and organization of these compartments. In parallel, I will use mass spectrometry to reveal the constituents of these assemblies and subsequently interrogate the functional roles of these constituents in supporting phage infection. From my multidisciplinary studies, I stand to uncover fundamentally new phage biology in addition to innovative and potentially transferable mechanisms to enhance phage success in the fight against pathogenic bacteria. I will perform this work at UCSF, an institute that hosts state-of-the-art research facilities and fosters an intellectually rigorous and collaborative research environment. Co-supervision in the Bondy-Denomy and Agard labs will ensure thorough training in phage biology, genetics and biophysics. Through the course of my research, I will undergo extensive training and subsequently gain expertise in diverse fields of microbiology, molecular biology, biophysics and structural biology that I need to fulfill my postdoctoral training goals and subsequently pioneer an independent research program.