RNA penetrations into bacteria.

RNA 渗透到细菌中。

• 批准号: 10445162
• 负责人: Lanying Zeng
• 金额: $ 46.32万
• 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/10445162
• 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; 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; Microscopy; Modeling; Molecular; Multi-Drug Resistance; Pathway interactions; Penetration; Pilum; Plasmids; Play; Process; Proteins; Pseudomonas Infections; Pseudomonas aeruginosa; RNA; RNA Phages; RNA delivery; Regulation; Resistance; Resolution; Role; Site-Directed Mutagenesis; Structure; Testing; Toxin; Virion; Virulence; Virulence Factors; Virus; Work; base; 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; success; 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.

项目概要 单链 RNA 噬菌体（ssRNA 噬菌体）是小型近二十面体病毒， 利用RNA作为遗传物质，通过回缩菌毛来感染细菌。最近超过 15,000 个新 ssRNA 噬菌体已被鉴定，但其宿主和感染机制仍不清楚。的 ssRNA噬菌体感染周期中的步骤，噬菌体如何包装基因组RNA以及 识别其特定宿主仅已知模型 ssRNA 大肠杆菌噬菌体，例如 MS2 和 Qβ；和 RNA 最终如何传递到细胞质中尚不清楚。从初步数据来看，PI 发现之前基于模型为ssRNA噬菌体感染机制设定的范式 大肠杆菌噬菌体不能再应用于其他 ssRNA 噬菌体。 ssRNA 噬菌体的宿主受体，即回缩菌毛，通常与噬菌体的毒力有关。 致病菌和抗生素抗性质粒的共享。该项目将重点关注 噬菌体 PP7 和 AP205，感染铜绿假单胞菌和不动杆菌属， 分别通过 IV 型菌毛 (T4P)。总体目标是确定所涉及的机制 PP7/AP205 包装以及 RNA 渗透到宿主中，该过程涉及宿主 识别和RNA进入。具体目标是揭示（1​​）的分子机制 PP7/AP205 的包装，(2) RNA 进入之前 PP7/AP205 和 T4P 之间的相互作用，以及 (3)RNA进入过程中T4P的脱离。这项工作不仅揭示了对 ssRNA 噬菌体的感染机制，同时也为设计 ssRNA 噬菌体提供指导 目的如下：ssRNA噬菌体将被设计为分离致病菌菌毛的手段 细菌，作为治疗多重耐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传播 通过破坏菌毛而使细胞失活，同时让细胞生长，而不会对细胞施加选择压力 宿主产生进一步的抵抗力。这种方法还避免释放任何不需要的 细胞内容物，包括 DNA、蛋白质和毒素进入环境，可能会干扰 其他细菌或影响人体细胞。未来，拟建项目也将为 开发一种将大量外源 RNA 包装和递送到体内的方法 细菌细胞。由于细胞内 RNA 的寿命很短，它们可以瞬时调节 细胞，并且不太可能像 DNA 质粒那样发挥长期遗传效应。在 此外，使用 ssRNA 噬菌体进行 RNA 递送不依赖于细胞的人工制备 能够进行热休克或电穿孔，这在原位很难进行。