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

动物肠道中有一个复杂的特殊适应性细菌群落，这些细菌直接与宿主上皮细胞接触，并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起着至关重要的作用，因此详细了解细菌如何在肠道中定植以及生物和生物物理因素如何影响其与宿主共生存在的稳定性和寿命具有重大意义。 一个经常被忽视的因素可能在构建肠道相关细菌群落中发挥重要作用，那就是病毒的存在，特别是噬菌体（感染细菌的病毒）。研究人员开发了一种简单的海洋无脊椎动物模型系统（海鞘Ciona Ciona），该系统提供了解剖宿主细菌共生的许多方面的机会，例如初始定植，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，他们将测试这样一个假设，即细菌通过分解特定宿主的能力在塑造整体细菌群落结构方面发挥重要作用，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。通过仔细研究体外和海鞘肠道内的噬菌体-细菌相互作用，他们将获得前所未有的洞察力，了解细菌对动物肠道内细菌群落的影响。这项研究将有益于社会，探索微生物重塑复杂微生物群落的前景，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推广计划，鼓励高中生开展包括微生物生态学概念在内的指导性科学博览会项目，并提供分子微生物学技术的实践培训。为了达到明确的目标受众，他们还将运行微生物生态学模块的女童子军，女孩公司，和南佛罗里达大学的海洋学营的女孩。为了研究细菌在塑造细菌群落的整体结构和功能的作用，研究人员将完成三个具体的研究目标：1）使用基于培养和不依赖培养的方法表征玻璃海鞘肠道中的噬菌体多样性，2）在体外解剖噬菌体-细菌相互作用，并确定噬菌体是否可以改变细菌群落（微生物组）结构，和3）在水中存在和不存在细菌的情况下培养玻璃海鞘以确定噬菌体存在对肠道微生物组和宿主生理学的影响。 作为一种滤食性固着生物，玻璃海鞘直接与海洋环境接触，并遇到连续不断的微生物，这些微生物可能会定植或影响其肠道植物群。 以前对成年人的研究，现场收获，Ciona表明，一致的“核心”微生物组主导着他们的肠道。此外，他们已经成功培养了核心肠道微生物组的各种成员，分离了其中一些细菌的细菌，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，这些基质类似于存在和不存在细菌的肠道上皮细胞的表面。 玻璃海鞘是一个非常容易处理的实验系统，可以在无菌条件下生长，允许实验控制宿主，细菌和寄生虫之间共生对话的几乎每个方面。 这项工作的结果具有变革性的潜力，可以重新定义我们对建立和维持稳定的宿主相关细菌群落的因素的理解，并反过来影响宿主本身的生理学。

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.