Collaborative Research: Infection mechanisms of bacteriophages targeting motile bacteria

噬菌体是感染细菌的病毒。一个成功的噬菌体必须遇到、识别和进入一种称为宿主的特定类型的细菌。然后，它接管宿主繁殖机器，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副本。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鲜为人知的组成部分。一组专门针对可移动细菌的噬菌体。这些噬菌体与细菌鞭毛结合，作为接触和感染宿主细胞的一种手段。病毒颗粒被认为沿着旋转的细菌鞭毛表面迁移，到达细胞体进行感染。然而，对噬菌体移位和随后的感染过程中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，以及噬菌体DNA进入。结果将改变我们目前对依赖鞭毛的噬菌体生物学概念及其对特定宿主的适应。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将噬菌体作为“抗生素”进行治疗。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，特别是代表不足的人群和女性。公共推广活动包括在校园内外为小学生和高中生进行动手演示，并让本科生和高中生参与研究。嗜鞭毛噬菌体通过搭便车在其旋转的鞭毛上进行感染，以攻击可移动的细菌。除了缺乏对噬菌体运动的直接可视化外，在其宿主的鞭毛上吸附和移位后的感染过程，特别是与细胞表面的结合和DNA进入，是未知的。该研究项目的目的是阐明这组专门的噬菌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。两个嗜鞭毛噬菌体和它们的细菌宿主将作为模式系统：噬菌体7-7-1和CHI与宿主农杆菌。H13-3和大肠埃希菌/沙门氏菌分别为鼠伤寒沙门氏菌。两种寄主都有不同的鞭毛系统：农杆菌鞭毛只顺时针旋转，更坚硬，以适应更粘稠的土壤环境，而肠杆菌的鞭毛改变旋转方向，更灵活。首先，研究人员将表征噬菌体的吸附特性，以接触和感染它们的细菌宿主。将通过蛋白质-蛋白质相互作用实验、质谱学和突变分析来揭示噬菌体-鞭毛相互作用的分子基础。其次，将在原子尺度上进行最先进的微流控成像，并结合原位透射电子显微镜，实时描绘噬菌体沿鞭毛转移的机制。第三，将使用遗传和酶技术来表征导致DNA进入的噬菌体结合事件，并将使用超分辨荧光显微镜来检测噬菌体DNA的命运。这项拟议的研究将促进对依赖鞭毛的噬菌体感染机制的了解。这项研究是由整合和组织系统部门的共生、感染和免疫项目以及生物科学理事会分子和细胞生物科学部门的细胞动力学和功能集群共同资助的。这一奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.