RNA penetrations into bacteria.

RNA 渗透到细菌中。

• 批准号: 10674975
• 负责人: Lanying Zeng
• 金额: $ 46.96万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10674975
• 关键词: Acinetobacter; Adsorption; Affect; Antibiotic Resistance; Bacteria; Bacterial Infections; Bacterial Pili; Bacteriophages; Binding; Binding Proteins; Biology; Capsid; Capsid Proteins; Cells; Coliphages; Color; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cytosol; DNA; DNA delivery; Data; Data Set; Dedications; Electroporation; Engineering; Enterobacteria phage MS2; Environment; Escherichia coli; Fluorescence Microscopy; Future; Genetic; Genetic Materials; Goals; Gram-Negative Bacteria; Guidelines; Health Care Costs; Heat-Shock Response; Human; Image; In Situ; Infection; Knowledge; Label; Life; Measures; Methods; Modeling; Molecular; Multi-Drug Resistance; Pathway interactions; Penetration; Pilum; Plasmids; Play; Process; Proteins; Pseudomonas; Pseudomonas aeruginosa; RNA; RNA Phages; RNA delivery; Regulation; Resistance; Resolution; Role; Site-Directed Mutagenesis; Structure; Testing; Toxin; Traction; Virion; Virulence; Virulence Factors; Virus; Visualization; Work; cell preparation; ds-DNA; experimental study; fluorescence imaging; genomic RNA; in situ imaging; insight; interest; kinetosome; member; multi-scale modeling; mutant; pathogen; pathogenic bacteria; plasmid DNA; pressure; receptor; structural determinants; success; superresolution microscopy; time use; tool

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.

项目摘要 单链RNA细菌（ssRNA噬菌体）是小的近牙卷病毒， 使用RNA作为遗传物质，通过缩回pili感染细菌。最近> 15,000个新的ssrna 已经确定了噬菌体，但是他们的宿主和感染机制仍然未知。的 ssRNA噬菌体感染周期期间的步骤，噬菌体如何包装基因组RNA和 识别其特定宿主仅在模型ssRNA coliphages（例如MS2和Qβ）中闻名。和 最终如何将RNA传递到细胞质中是晦涩的。从初步数据中，PI 发现先前的范式设置了基于模型的SSRNA噬菌体感染机制 Coliphages不能再应用于其他SSRNA噬菌体。 ssRNA噬菌体的宿主受体，可伸缩的pili，通常参与 致病细菌和抗生素耐药的等离子酰胺的共享。这个项目将重点放在 Phages PP7和AP205，感染了铜绿假单胞菌和ACINETOBACTER spp。 分别通过IV型Pili（T4P）。总体目标是确定所涉及的机制 在PP7/AP205包装中，RNA渗透到宿主中，这两个过程都涉及两个主机 识别和RNA进入。具体目的是揭示（1​​）的分子机制 PP7/AP205的包装，（2）RNA进入前PP7/AP205和T4P之间的相互作用，以及 （3）RNA进入过程中T4P的脱离。这项工作不仅会揭示有关的见解 ssRNA噬菌体的感染机制，但也为工程师ssRNA噬菌体提供指南 以下目的：ssRNA噬菌体将被设计为脱离致病性的手段 细菌，作为治疗多药耐药细菌感染的替代策略。与众不同 通过裂解病原体，病毒和抗生素耐药性传播传统的噬菌体疗法是 通过在离开细胞生长的同时破坏Pili，而无需在不施加选择性压力的情况下灭活 宿主发展进一步的阻力。这样的方法还避免了任何不需要的 细胞含量包括DNA，蛋白质和毒素进入环境，可能会干扰 其他细菌或影响人类细胞。将来，拟议的项目还将提供基础 用于开发一种将大量外国RNA包装和交付的方法 细菌细胞。由于细胞内RNA的寿命短，它们允许对 像DNA质粒一样，细胞且不太可能发挥长期遗传作用。 此外，用ssRNA噬菌体递送RNA不依赖细胞的人工制备 有能力进行热震或电穿孔，这很难原地执行。