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，通过可伸缩的皮利感染细菌。最近> 15，000个新ssRNA 虽然已经发现了这种病毒，但它们的宿主和感染机制仍不清楚。的 在ssRNA病毒感染周期中的步骤，病毒如何包装基因组RNA， 识别其特异性宿主仅为模式ssRNA大肠杆菌所知，如MS 2和Qβ;以及 RNA最终是如何进入胞质溶胶的还不清楚。根据初步数据，PI 发现先前基于模型的ssRNA噬菌体感染机制的范例集 大肠杆菌素不能再应用于其他ssRNA酶。 ssRNA的宿主受体，即可伸缩的皮利，通常与细菌的毒力有关。 病原菌和耐药质粒的共享。该项目将重点关注 感染铜绿假单胞菌和不动杆菌属（Acinetobacter spp.） 分别通过IV型皮利（T4 P）。总体目标是确定所涉及的机制 在PP 7/AP 205包装中，RNA渗透进入宿主，这一过程既涉及宿主 识别和RNA进入。具体的目的是揭示分子机制（1） PP 7/AP 205的包装，（2）RNA进入前PP 7/AP 205和T4 P之间的相互作用，和 (3)RNA进入过程中T4 P的脱离。这项工作不仅将揭示洞察 而且还提供了工程化ssRNA酶指导， 以下目的：ssRNA微球将被工程化作为分离致病菌的皮利的手段， 细菌，作为治疗多重耐药细菌感染的替代策略。不像 通过裂解病原体、毒力和抗生素耐药性传播的传统噬菌体疗法， 通过破坏皮利而使细胞生长而灭活，而不对细胞施加选择性压力。 宿主产生进一步的抵抗力这种方法还避免了任何不需要的释放。 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。今后，该项目也将提供基础 用于开发将大量外源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内的蛋白质。 细胞，不太可能像DNA质粒那样产生长期的遗传效应。在 此外，使用ssRNA引物的RNA递送不依赖于细胞的人工制备 能胜任热休克或电穿孔，这是很难在原位进行。