Uncovering the roles of phages in the ecology of Porphyromonas gingivalis in periodontal disease

项目摘要/摘要 拟议工作的广泛目标是解决对细菌病毒作用缺乏了解的问题 (噬菌体)在塑造口腔微生物群落生态方面的作用。噬菌体是重要的捕食者 细菌在所有环境中的共生体，它们在口腔中含量丰富(唾液中高达108毫升-1 和107 mg-1斑块)。通过对细菌宿主的急性和慢性感染，噬菌体具有 影响细菌种群结构、定植动态和菌株水平变异的可能性 致命性。然而，尽管口腔微生物群中的细菌已经被研究了几十年，但人们对此知之甚少。 噬菌体如何塑造这些群落的结构和功能，从而在人类中发挥作用 健康和疾病。在这项应用中，一种细菌病原体一直-并将继续- 深入研究它在牙周病--卟啉单胞菌中的关键物种和驱动因素 牙周炎(Pg)是一种研究口腔微生物群中细菌与噬菌体相互作用的模型。至 到目前为止，还没有分离出能够感染或杀死PG的噬菌体，也没有系统地研究PG基因组 被调查以寻找长期感染形式的噬菌体(“预言体”)的证据。的中心假说 拟议的工作是PG分离株拥有不同的噬菌体，这些噬菌体整合到它们的基因组中作为前驱噬菌体， 这些前驱体有可能改变口腔微生物群中PG的生态。二 相辅相成的目标，提供了对噬菌体在生态中的作用的广度和深度的洞察 PG，用于解决这一假说。在目标1中，目标是确定和确定功能特征 Pg基因组中编码的前噬菌体。这是通过从志愿者身上获得新的PG分离株来实现的 利用牙周病来扩大可用的PG基因组的数量，并通过生物信息识别 以及描述这些基因组中的先知噬菌体，包括它们对 细菌毒力和种内竞争。在目标2中，目标是确定 预言体感染它们的PG宿主。这是通过建立PG噬菌体的模型系统和使用 基于栽培的方法来确定它们的寄主范围决定因素。完成这些目标将提供 对PG噬菌体系统发育和功能多样性的第一次观察，对其寄主范围的洞察，以及 首次鉴定出它们用来感染PG宿主的受体。这将为研究提供 具有检验假说所需的知识库和工具的社区(体外、体内和体内 纵向和横向研究)，这些研究解决了与噬菌体的相互作用如何塑造PG基因组， 生理学、种群动态、物种间和王国间的相互作用以及在牙周中的作用 疾病及其发展。揭示噬菌体-细菌相互作用的基本原理 口腔微生物组的结构也将为合理设计健壮、安全、 以及高效的基于噬菌体的疗法，用于口腔和全身疾病，包括牙周病。

PROJECT SUMMARY/ABSTRACT The broad goal of the proposed work is to address the lack of knowledge about the roles of bacterial viruses (phages) in shaping the ecology of oral microbial communities. Phages are important predators and symbionts of bacteria in all environments, and they are abundant in the oral cavity (up to 108 ml-1 in saliva and 107 mg-1 plaque). Through acute and chronic infections of their bacterial hosts, phages have the potential to shape bacterial population structure, colonization dynamics, and strain level variation in virulence. Yet, though bacteria in the oral microbiome have been studied for decades, little is known about how phages shape the structure and function of these communities and thereby may play a role in human health and disease. In this application, a bacterial pathogen that has been – and continues to be - intensively studied for its role as keystone species and driver in periodontal disease, Porphyromonas gingivalis (Pg), is developed as a model for studying bacteria-phage interactions in the oral microbiome. To date, no phages able to infect or kill Pg have been isolated, nor have Pg genomes been systematically investigated for evidence of chronically infecting forms of phages (“prophages”). The central hypothesis of the proposed work is that Pg isolates harbor diverse phages integrated into their genomes as prophages, and that these prophages have the potential to alter the ecology of Pg in the oral microbiome. Two complementary aims, providing both breadth and depth of insight into the roles of phages in the ecology of Pg, are used to address this hypothesis. In Aim 1, the goal is to identify and functionally characterize prophages encoded in genomes of Pg. This is achieved by obtaining new isolates of Pg from volunteers with periodontal disease to expand the number of available Pg genomes, and bioinformatically identifying and characterizing prophages in these genomes, including with respect to their capacity to contribute to bacterial virulence and intraspecies competition. In Aim 2, the goal is to determine the receptors used by prophages to infect their Pg hosts. This is achieved by establishing model systems of Pg phages and using cultivation based methods to identify their host range determinants. Completion of these aims will provide the first view of the phylogenetic and functional diversity of Pg phages, insights into their host ranges, and the first identification of receptors that they use to infect their Pg hosts. This will provide the research community with the knowledgebase and tools necessary to test hypotheses (in vitro, in vivo, and in longitudinal and cross-sectional studies) that address how interactions with phages shape Pg genomes, physiology, population dynamics, inter-species and inter-kingdom interactions, and role in periodontal disease and its progression. Uncovering fundamental principles of how phage-bacteria interactions are structured in the oral microbiome will also lay an essential foundation for the rational design of robust, safe, and efficient phage-based therapeutics for use in oral and systemic disease including periodontal disease.