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。成功的噬菌体必须遇到、识别并进入一种称为宿主的特定类型的细菌。然后，它接管宿主的生殖机制来复制遗传物质、DNA，并在杀死宿主细胞之前复制更多的噬菌体。噬菌体非常丰富，对微生物种群具有重大影响，但它们是任何生态系统中人们最了解的组成部分。一组专门的噬菌体以运动细菌为目标。这些噬菌体与细菌鞭毛结合，作为接触和感染宿主细胞的手段。人们认为病毒颗粒沿着旋转的细菌鞭毛表面迁移到达细胞体进行感染。然而，人们对噬菌体易位和随后的感染过程所涉及的具体过程知之甚少。该项目的总体目标是使用两个噬菌体-细菌模型系统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。结果将改变我们目前对鞭毛依赖性噬菌体及其对特定宿主的适应的生物学概念。这项研究的结果可以通过利用噬菌体将噬菌体用作农业和临床环境中的“抗生素”来直接有益于未来的农业和环境问题。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群和女性。公共宣传活动包括在校内和校外为小学生和高中生进行实践演示，以及让本科生和高中生参与研究。嗜鞭毛噬菌体通过搭乘旋转鞭毛进行感染来瞄准运动细菌。除了缺乏噬菌体运动的直接可视化之外，宿主鞭毛吸附和易位后的感染过程，特别是与细胞表面的结合和 DNA 进入，也是未知的。该研究项目的目的是阐明这一特殊噬菌体群体利用鞭毛动力学和随后的细胞表面相互作用来支持噬菌体有效繁殖的逐步过程。两种嗜鞭毛噬菌体及其细菌宿主将作为模型系统：噬菌体 7-7-1 和 Chi 以及宿主农杆菌。分别为 H13-3 和大肠杆菌/肠沙门氏菌鼠伤寒血清型。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，并且为了适应更粘稠的土壤环境而更加刚性，而肠杆菌的鞭毛则改变旋转方向并且更加灵活。首先，研究人员将表征噬菌体接触和感染细菌宿主的吸附特性。蛋白质-蛋白质相互作用实验、质谱分析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，将进行最先进的原子尺度微流体成像，与原位透射电子显微镜相结合，以实时描绘噬菌体沿鞭毛易位的机制。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，并使用超分辨率荧光显微镜来检查噬菌体 DNA 的命运。拟议的研究将增进对鞭毛依赖性噬菌体感染机制的了解。这项研究由生物科学理事会综合和有机系统司的共生、感染和免疫计划以及分子和细胞生物科学司的细胞动力学和功能集群共同资助。该奖项反映了 NSF 的法定使命，并被认为值得支持 通过使用基金会的智力优点和更广泛的影响审查标准进行评估。