Role of Phages in Structuring Bacterial Communities: Ciona Intestinalis as a Model Organism

动物的肠道中有一个复杂的特殊适应细菌群落，这些细菌直接与宿主上皮细胞接触，从根本上影响宿主的健康和营养。由于肠道细菌在宿主健康中扮演着如此重要的角色，详细了解细菌如何在肠道中定植，以及生物学和生物物理因素如何影响它们与宿主共生的稳定性和持久性具有巨大的意义。在构建肠道相关细菌群落中可能发挥重要作用的一个经常被忽视的因素是病毒的存在，特别是噬菌体(感染细菌的病毒)的存在。研究人员开发了一个简单的海洋无脊椎动物模型系统(海鞘)，该系统提供了剖析宿主的许多方面的机会：细菌共生，如初始定殖、摄食和消化的影响，以及与宿主免疫系统的相互作用。在这个项目中，他们将检验这一假设，即噬菌体通过分解特定宿主的能力，从而释放溶解的有机物，促进宿主之间的基因转移，并推动微生物进化，从而在塑造整体细菌群落结构方面发挥重要作用。通过仔细研究噬菌体与细菌在体外和海鞘肠道内的相互作用，他们将前所未有地深入了解噬菌体对动物肠道内细菌群落的影响。这项研究将通过探索噬菌体重塑复杂微生物群落的前景来造福社会，这对人类健康、生物修复和生态系统恢复项目具有重要意义。研究人员将制定一项外展计划，鼓励高中生开展包含微生物生态学概念的指导性科学博览会项目，并提供分子微生物学技术的动手培训。为了接触到不同的目标受众，他们还将为女童子军、女孩公司和南佛罗里达大学的女孩海洋夏令营运行微生物生态模块。为了检验噬菌体在塑造细菌群落整体结构和功能中的作用，研究人员将完成三个具体的研究目标：1)使用基于培养和非培养的方法表征Ciona肠道中的噬菌体多样性；2)体外解剖噬菌体与细菌的相互作用，并确定噬菌体是否可以改变实验定居的Ciona中的细菌群落(微生物群)结构，3)在水中有无噬菌体的情况下培养乔纳，以确定噬菌体的存在对肠道微生物群落和宿主生理的影响。作为一种滤食性固着生物，Ciona直接与海洋环境接触，并遇到持续不断的微生物流，这些微生物可能会定居或影响其肠道菌群。西奥纳之前对成年的田间收获的研究表明，它们的肠道中有一个一致的“核心”微生物群。此外，他们还成功地培养了核心肠道微生物组的各种成员，为其中一些细菌分离了噬菌体，并正在研究这些细菌在人工构建的基质上形成细胞外生物膜的过程，这些基质类似于肠道上皮的表面，在存在和不存在噬菌体的情况下。Ciona是一种高度可处理的实验系统，可以在主要无菌条件下生长，允许对宿主、细菌和噬菌体之间共生对话的几乎每个方面进行实验控制。这项工作的结果有可能重新定义我们对影响稳定的、与宿主相关的细菌群落的建立和维持并进而影响宿主本身的生理的因素的理解。

The animal gut hosts a complex community of specially-adapted bacteria that interface directly with the host epithelial cells and fundamentally affect host health and nutrition. Because gut bacteria play such vital roles in host health, a detailed understanding of how bacteria colonize the gut and how biological and biophysical factors affect the stability and longevity of their symbiotic existence with the host is of enormous significance. One often overlooked factor that may play a significant role in structuring gut-associated bacterial communities is the presence of viruses, specifically phages (viruses that infect bacteria). The investigators have developed a simple marine invertebrate model system (the sea squirt Ciona intestinalis) that affords the opportunity to dissect numerous aspects of host:bacteria symbiosis, such as initial colonization, effects of feeding and digestion, and interactions with the host immune system. In this project, they will test the hypothesis that phages serve important roles in shaping overall bacterial community structure through their ability to break apart specific hosts, thereby releasing dissolved organics and facilitating gene transfer among hosts, and driving microbial evolution. By carefully examining phage-bacteria interactions both in vitro and within the gut of the sea squirt, they will gain unprecedented insight into the effects of phages on bacterial communities living within animal guts. This research will benefit society by exploring the promise of phages for remodeling complex microbial communities, which has important implications for human health, bioremediation, and ecosystem restoration projects. The investigators will develop an outreach program that encourages high school students to undertake mentored science fair projects encompassing microbial ecology concepts and provide hands-on training in molecular microbiology techniques. To reach a distinct target audience, they will also run microbial ecology modules for Girl Scouts, Girls Incorporated, and the University of South Florida's Oceanography Camp for Girls.To examine the roles of phages in shaping the overall structure and function of bacterial communities, the investigators will accomplish three specific research objectives: 1) characterize phage diversity in the Ciona gut using culture-based and culture independent methods, 2) dissect phage-bacteria interactions in vitro and determine if phages can modify bacterial community (microbiome) structure in experimentally colonized Ciona, and 3) culture Ciona in both the presence and absence of phages in the water to determine the effects of phage presence on gut microbiomes and host physiology. As a filter-feeding sessile organism, Ciona directly interfaces with the marine environment and encounters a continuous stream of microbes that can potentially colonize or influence its gut flora. Previous work with adult, field-harvested, Ciona indicated that a consistent, "core" microbiome dominates their gut. Additionally, they have successfully cultured various members of the core gut microbiome, isolated phages for some of these bacteria, and are studying the process of extracellular biofilm formation by these bacteria on artificially constructed matrices that resemble the surface of the gut epithelium in the presence and absence of phages. Ciona is a highly tractable experimental system that can be grown under predominantly sterile conditions, allowing experimental control of of nearly every aspect of the symbiotic conversation between host, bacteria, and phages. The results of this work have the transformative potential of redefining our understanding of the factors that shape the establishment and maintenance of stable, host-associated, bacterial communities and in turn affect the physiology of the host itself.