RNA penetrations into bacteria.

项目摘要 单链RNA噬菌体(ssRNA噬菌体)是一种近二十面体的小病毒， 利用RNA作为遗传物质，通过可伸缩的菌毛感染细菌。最近&gt；15,000个新的单链RNA 噬菌体已被鉴定，但其宿主和感染机制尚不清楚。中的 在单链RNA噬菌体感染周期中，噬菌体如何包装基因组RNA和 识别其特定宿主仅对模型单链RNA噬菌体已知，如MS2和Qβ；以及 RNA最终是如何输送到细胞质中的，目前尚不清楚。从初步数据来看，PI 发现以往的范式集为基于模型的单链RNA噬菌体感染机制 大肠杆菌噬菌体不能再应用于其他单链RNA噬菌体。 单链RNA噬菌体的宿主受体，即可伸缩的菌毛，通常与沙门氏菌的毒力有关 病原菌和耐药质粒的共享。该项目将重点放在 感染铜绿假单胞菌和不动杆菌的噬菌体PP7和AP205， 分别通过IV型菌毛(T4P)。总体目标是确定所涉及的机制 在PP7/AP205的包装中，RNA渗透到主机中，这是一个涉及主机和主机的过程 识别和RNA输入。具体目的是揭示(1)的分子机制 PP7/AP205的包装，(2)在RNA进入之前PP7/AP205和T4P之间的相互作用，以及 (3)T4P在RNA进入过程中的脱落。这项工作不仅将揭示 单链RNA噬菌体的感染机制，也为工程单链RNA噬菌体提供了指导 目的：将单链RNA噬菌体作为分离致病菌菌毛的手段 细菌，作为治疗耐多药细菌感染的替代策略。不像 传统的噬菌体疗法通过裂解病原体，毒力和抗生素耐药性的传播 通过破坏菌毛来灭活，同时让细胞生长，而不对其施加选择性压力 宿主会进一步产生抵抗力。这样的方法还避免了释放任何不需要的 细胞内容物，包括DNA、蛋白质和环境中的毒素，可能会干扰 其他细菌或影响人类细胞。未来，拟议中的项目也将提供基础 为了开发一种将大量外来RNA包装和递送到 细菌细胞。由于RNA在细胞内的寿命很短，它们允许瞬时调节 细胞，而且不太可能像DNA质粒那样产生长期的遗传效应。在……里面 此外，用单链核糖核酸噬菌体运送核糖核酸不需要人工制备细胞。 能承受热冲击或电穿孔，这很难在现场进行。

Project Summary Single-stranded RNA bacteriophages (ssRNA phages) are small near-icosahedral viruses that use RNA as genetic material to infect bacteria through retractile pili. Recently >15,000 new ssRNA phages have been identified but their hosts and mechanisms of infection remain unknown. Of the steps during the infection cycle of ssRNA phages, how phages package the genomic RNA and recognize its specific host are only known for model ssRNA coliphages such as MS2 and Qβ; and how RNA is ultimately delivered into the cytosol is obscure. From the preliminary data, the PIs find that the previous paradigm set for the infection mechanism of ssRNA phage based on model coliphages can no longer be applied to other ssRNA phages. Host receptors of ssRNA phages, the retractile pili, are usually involved in the virulence of pathogenic bacteria and the sharing of antibiotic-resistant plasmids. This project will focus on phages PP7 and AP205, which infect Pseudomonas aeruginosa and Acinetobacter spp., respectively, via the Type IV pili (T4P). The overall goal is to determine the mechanisms involved in PP7/AP205 packaging, and RNA penetration into the host, a process which involves both host recognition and RNA entry. Specific aims are to reveal the molecular mechanisms for (1) the packaging of PP7/AP205, (2) the interplay between PP7/AP205 and T4P before RNA entry, and (3) the detachment of T4P during RNA entry. This work will not only reveal insights into the infection mechanism of ssRNA phages but also provide guidelines to engineer ssRNA phages for the following purpose: ssRNA phages will be engineered as means to detach pili of pathogenic bacteria, as an alternative strategy for treating multidrug-resistant bacterial infections. Unlike traditional phage therapy by lysing pathogens, virulence and antibiotic resistance spread are inactivated by breaking pili while leaving the cells to grow, without exerting selective pressure on the host to develop further resistance. Such a method also avoids the release of any unwanted cell contents including DNA, proteins, and toxins into the environment which could interfere with other bacteria or affect human cells. In the future, the proposed project will also provide a basis for developing a method for packaging and delivery of a large number of foreign RNAs into bacterial cells. Due to the short life of RNAs inside the cell, they allow transient regulation of the cells and are less likely to exert long-term genetic effects as in the case of DNA plasmids. In addition, RNA delivery with ssRNA phages does not rely on the artificial preparation of cells competent for heat-shock or electroporation, which is hard to perform in situ.