Elucidation of the mechanisms of jumbophage genome protection during infection

面对病原菌对现有大多数抗生素产生抗药性的公共卫生危机，噬菌体 (细菌病毒；噬菌体)自然感染和杀死细菌的，是一种很有前途的替代品 抗菌剂。然而，实现这一翻译目标的一个重要障碍是过多的 细菌中抵抗噬菌体感染的免疫机制。克服这一障碍需要噬菌体 具有强大的抗免疫能力，可杀死多个抗药性病原菌菌株。 在这方面，ΦKZ样噬菌体(带有基因组的噬菌体；200kb)作为一个家族出现，具有杰出的 在感染和众多家庭中挫败大量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。巨噬菌体ΦKZ是一种寄主范围很广的 多重耐药铜绿假单胞菌的杀手及主导模式 巨噬菌体家族的噬菌体。免疫逃避很大程度上是通过组装 噬菌体编码的蛋白核样“壳”隔间，包裹和屏蔽复制 噬菌体基因组。然而，在外壳组装之前，这种噬菌体是如何保护其基因组的，目前尚不清楚。 这项提议的长期目标是了解用来保护被喷出的基因组的机制 ФKZ样噬菌体-初步证据(来自我们和其他人)表明一种大分子组装 在感染细胞中与被喷出的巨噬菌体基因组位置相似的隔室(在外壳组装之前)。 目前，这些络合物的组成和生物学意义尚不清楚。我假设 这种复合体(在这里被称为“喷射结构”)产生了一个含有DNA的细胞器，它包裹着 保护排出的噬菌体基因组不受免疫核酸酶的影响。我将首先使用荧光显微镜和 冷冻电子断层扫描，以可视化贝壳组装前的感染状态。从这些研究中，我将获得 对这些隔室的生物发生和组织的高分辨率结构洞察。同时，我会 使用质谱学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，我认为 除了创新和潜在的可移植机制外，还将从根本上发现新的噬菌体生物学 以增强噬菌体在对抗病原体方面的成功。 我将在加州大学旧金山分校执行这项工作，这是一个拥有最先进研究设施的研究所，并培养 智力严谨和协作的研究环境。《邦迪正统》中的联合监管和 阿加德实验室将确保在噬菌体生物学、遗传学和生物物理学方面进行全面培训。在我的人生历程中 在研究方面，我将接受广泛的培训，随后获得不同领域的微生物学专业知识， 分子生物学，生物物理学和结构生物学，我需要完成我的博士后培训目标和 随后开创了一项独立的研究计划。

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.