Collaborative Research: Infection mechanisms of bacteriophages targeting motile bacteria

合作研究：噬菌体针对运动细菌的感染机制

• 批准号: 2054392
• 负责人: Birgit Scharf
• 金额: $ 87.28万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2054392&HistoricalAwards=false
• 关键词: Collaborative; Research; Infection; mechanisms; bacteriophages

Bacteriophages are viruses that infect bacteria. A successful phage must encounter, recognize, and enter a specific type of bacteria, called a host. Then, it takes over the host reproductive machinery to replicate the genetic material, DNA, and make more phage copies before killing the host cell. Phages are highly abundant and have a major influence on microbial populations, but they are the most poorly understood components in any ecosystem. A specialized group of bacteriophages targets motile bacteria. These phages bind to the bacterial flagellum as a means of contacting and infecting host cells. Viral particles are thought to migrate along the surface of rotating bacterial flagella to reach the cell body for infection. However, little is known about the specific processes involved in phage translocation and subsequent infection processes. The overarching goal of this project is to directly visualize phage movement, analyze cell surface binding, and phage DNA entry using two phage-bacteria model systems. Outcomes will transform our current concepts of the biology of flagella-dependent phages and their adaptation to specific hosts. The results of this research can directly benefit future agricultural and environmental issues by harnessing bacteriophages for the therapeutic use of phages as “antibiotics” in agriculture and clinical contexts. Broader Impacts activities will involve the interdisciplinary training of graduate students. The team is committed to mentoring graduate and undergraduate students, especially underrepresented populations and women. Public outreach activities include hands-on demonstrations for elementary and high school students, both on- and off-campus, and involvement of undergraduate and high school students in research.Flagellotropic phages target motile bacteria by hitchhiking on their rotating flagella for infection. In addition to a lack of direct visualization of phage movement, the infection processes following adsorption to and translocation on its host’s flagella, particularly binding to the cell surface and DNA entry, are unknown. The aim of the research project is to elucidate the stepwise processes by which this specialized group of phages uses flagella dynamics and subsequent cell surface interactions to support effective phage propagation. Two flagellotropic phages and their bacterial hosts will serve as model systems: phage 7-7-1 and Chi with hosts Agrobacterium sp. H13-3 and Escherichia coli/Salmonella enterica serovar Typhimurium, respectively. Both hosts possess divergent flagellar systems: Agrobacterium flagella rotate only clockwise and are more rigid as adaptations to the more viscous soil environment while the flagella of enterobacteria switch rotational direction and are more flexible. First, the investigators will characterize the adsorption properties of phages to engage and infect their bacterial hosts. Protein-protein interactions experiments, mass spectrometry, and mutational analyses will be used to uncover the molecular basis for phage-flagella interaction. Second, state-of-the-art microfluidic imaging at the atomic scale, in conjunction with in situ transmission electron microscopy, will be performed to delineate the mechanism of phage translocation along the flagellum in real time. Third, genetic and enzymatic techniques will be employed to characterize phage binding events that lead to DNA entry and super-resolution fluorescence microscopy will be performed to examine the fate of phage DNA. The proposed research will advance knowledge of the infection mechanisms of flagella-dependent bacteriophages.This research is co-funded by the Symbiosis, Infection, and Immunity program in the Division of Integrative and Organismal Systems and the Cellular Dynamics and Function cluster in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

噬菌体是感染细菌的病毒。一个成功的噬菌体必须遇到、识别并进入一种特定类型的细菌，称为宿主。然后，它接管宿主的生殖机制，复制遗传物质，DNA，并在杀死宿主细胞之前制造更多的噬菌体拷贝。噬菌体非常丰富，对微生物种群有重大影响，但它们是任何生态系统中最不了解的组成部分。一组专门针对运动细菌的噬菌体。这些鞭毛与细菌鞭毛结合，作为接触和感染宿主细胞的手段。病毒颗粒被认为是沿着旋转的细菌鞭毛表面迁移到达细胞体进行感染。然而，很少有人知道的具体过程中涉及的噬菌体易位和随后的感染过程。该项目的首要目标是直接可视化噬菌体运动，分析细胞表面结合，和噬菌体DNA进入使用两个噬菌体-细菌模型系统。结果将改变我们目前的概念的生物学的鞭毛依赖性和适应特定的主机。这项研究的结果可以直接受益于未来的农业和环境问题，通过利用噬菌体在农业和临床环境中治疗性使用抗生素。更广泛的影响活动将涉及研究生的跨学科培训。该团队致力于指导研究生和本科生，特别是代表性不足的人口和妇女。公共推广活动包括为小学生和高中生在校内和校外进行的动手示范，以及大学生和高中生参与研究。鞭毛型细菌通过搭便车感染运动细菌。除了缺乏直接可视化的噬菌体运动，感染过程后吸附和易位在其宿主的鞭毛，特别是结合到细胞表面和DNA进入，是未知的。该研究项目的目的是阐明这一专门群体利用鞭毛动力学和随后的细胞表面相互作用来支持有效的噬菌体繁殖的逐步过程。两个鞭毛型噬菌体及其细菌宿主将作为模型系统：噬菌体7-7-1和Chi，分别与宿主土壤杆菌H13-3和大肠杆菌/沙门氏菌血清型鼠伤寒沙门氏菌。这两种宿主都具有不同的鞭毛系统：农杆菌鞭毛仅顺时针旋转，并且由于适应更粘稠的土壤环境而更加刚性，而肠杆菌的鞭毛切换旋转方向并且更加灵活。首先，研究人员将表征细菌的吸附特性，以吸引和感染它们的细菌宿主。蛋白质-蛋白质相互作用实验、质谱和突变分析将用于揭示噬菌体-鞭毛相互作用的分子基础。其次，国家的最先进的微流体成像在原子尺度上，结合原位透射电子显微镜，将进行描绘的机制，噬菌体易位沿着鞭毛在真实的时间。第三，将采用遗传和酶技术来表征导致DNA进入的噬菌体结合事件，并将进行超分辨率荧光显微镜检查噬菌体DNA的命运。这项拟议中的研究将推进鞭毛依赖性噬菌体感染机制的知识。这项研究是由共生，感染，该奖项反映了NSF的法定使命，并被认为值得支持通过使用基金会的知识价值和更广泛的影响审查标准进行评估。