Microbiome analysis of human oral bacteriophage communities

描述（由申请人提供）：通过微生物组研究对人类微生物群落的分析已经开始阐明构成人体的微生物的许多细节，以及它们对人类疾病和代谢过程的贡献。虽然微生物组分析的大部分焦点都集中在原核生物成分上，但对居住在人类环境中的病毒的关注相对较少。许多这样的病毒是专门感染细菌的噬菌体。噬菌体代表了地球上最丰富的感染因子，因为它们在细菌存在的任何地方都被发现，很可能在人体中有大量未被探索的噬菌体群落。通过它们杀死某些细菌的能力，或者通过引入新的基因编码功能来改造细菌，从而为这些细菌提供选择优势，噬菌体可能具有塑造人类细菌群落的深刻能力。虽然人们对噬菌体如何在体外与宿主细菌相互作用了解甚多，但对复杂的人类生态系统中这些相互作用的了解相对较少。提高我们对各种生态系统中噬菌体与宿主相互作用动力学的理解的一种方法是研究crispr （Clustered Regularly Interspaced Short Palindromic Repeats）。crispr代表了抵抗噬菌体捕食的适应性细菌免疫系统。它们的功能是将噬菌体DNA的一小部分（称为间隔段）结合在它们的细菌宿主基因组中的回文重复序列之间。间隔物掺入的结果是宿主细菌通过RNA干扰机制对先前遇到的噬菌体产生抗性。此外，通过间隔物的结合，CRISPR基因座可以被视为最近遇到的已产生耐药性的噬菌体的记录。此外，通过CRISPR机制对新遇到的噬菌体的耐药率可以通过对CRISPR含量随时间的采样来确定。目前还没有对人类口腔中噬菌体群落的全面研究。我们提出了一种结合分子生物学、微生物基因组学和生物信息学的多学科方法，以了解人类口腔噬菌体群落的性质，并了解对这些噬菌体产生耐药性的背后含义。我们汇编工作的短期目标是提高我们对人类口腔微生物群落动态的理解，并了解噬菌体对人类微生物群落的影响。长期目标是为学术医学的职业生涯奠定基础。本课题的具体目的如下：目的1：从人唾液中分离和表征噬菌体群体，确定细菌群落组成与噬菌体群落成员之间是否存在直接或反比关系。实验方法包括对不同人类受试者的噬菌体和细菌群落进行微生物组分析。目的2：评估人类受试者唾液中某些细菌的CRISPR含量，确定其获得唾液噬菌体对应间隔物的相对率。实验方法包括分析人类受试者某些口腔细菌中CRISPR位点含量随时间的变化，并将CRISPR位点含量与社区中噬菌体的存在或不存在进行比较。目的3：确定某些口腔细菌在体内是否对噬菌体捕食产生耐药性，并确定耐药性的机制是否由CRISPR介导。实验方法包括随着时间的推移从人类受试者中分离某些口腔细菌及其各自的噬菌体，检查噬菌体裂解其宿主细菌的能力，并评估CRISPR含量的变化。“人类口腔噬菌体群落的微生物组分析”PI: David T. Pride我们的研究计划包括提供人类口腔中噬菌体的第一个详细描述，以更好地了解居住在人体中的复杂微生物群落的动态。噬菌体具有改变原核生物群落的巨大能力，并且由于原核生物群落的扰动有助于疾病进程，因此对噬菌体的分析可能为我们人类受试者牙周炎和龋齿的发展提供重要线索。对疾病发展的影响使这项研究与公共卫生相关，并可能为开发用于诊断和预测与人类微生物群落有关的疾病的测试提供新的重点。

DESCRIPTION (provided by applicant): Analysis of human microbial communities through the study of microbiomes has begun to elucidate many details of both those organisms that constitute the human body, as well as their contribution to human disease and metabolic processes. While much of the focus of microbiome analysis has been on prokaryote constituents, there has been relatively little attention paid to viruses that inhabit the human environment. Many such viruses are bacteriophages that specifically infect bacteria. Bacteriophages represent the most abundant infectious agents on the planet, and because they have been found wherever bacteria exist, there likely are vast unexplored bacteriophage communities that inhabit the human body. Through either their ability to kill certain bacteria, or to transform bacteria by bringing in new gene-encoded function and thereby providing those bacteria a selective advantage, bacteriophages may have a profound ability to shape human bacterial communities. While much is known about the how bacteriophages interact with their host bacteria in vitro, there is relatively little known about these interactions within complex human ecosystems. One means for improving our understanding of the dynamics of bacteriophage interactions with their hosts in various ecosystems has been the study of CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPRs represent adaptive bacterial immune systems against phage predation. They function by incorporating small portions of bacteriophage DNA called spacers between palindromic repeats in the genome of their bacterial host. The result of spacer incorporation is resistance of the host bacterium to previously encountered bacteriophages through a mechanism of RNA interference. In addition, through the incorporation of spacers, the CRISPR locus could be viewed as a log of recently encountered bacteriophages to which resistance has developed. Furthermore, the rate of resistance to newly encountered bacteriophages through the CRISPR mechanism may be ascertained through sampling of CRISPR content over time. There have been no previous comprehensive studies of bacteriophage communities that inhabit the human oral cavity. We propose a multidisciplinary approach combining molecular biology, microbial genomics, and bioinformatics towards understanding the nature of human oral bacteriophage communities, and understanding the implications behind development of resistance to these bacteriophages. The short-term objectives of our compilation of work are to improve our understanding of human oral microbial community dynamics, and to understand the impact that bacteriophages have upon human microbial communities. The long-term objective is to lay a foundation for a career in academic medicine. Our specific aims for this proposal are as follows: Aim 1: To isolate and characterize bacteriophage populations from human saliva and determine whether there is a direct or inverse relationship between bacterial community composition and bacteriophage community members. The experimental approach involves microbiome analysis of bacteriophage and bacterial communities from different human subjects over time. Aim 2: To evaluate CRISPR content in certain salivary bacteria in human subjects and determine their relative rate of acquisition of spacers corresponding to salivary bacteriophages. The experimental approach involves analysis of changes in CRISPR loci content in certain oral bacteria in human subjects over time and comparison of CRISPR loci content with the presence or absence of bacteriophages in the community. Aim 3: To determine if certain oral bacteria develop resistance in vivo to bacteriophage predation, and ascertain whether the mechanism of resistance is CRISPR mediated. The experimental approach involves isolation of certain oral bacteria and their respective bacteriophages from human subjects over time, examining the ability of phage to lyse their host bacteria, and assessment of changes in CRISPR content. "Microbiome analysis of human oral bacteriophage communities" PI: David T. Pride Our research plan involves providing the first detailed description of bacteriophages in the human oral cavity to gain an improved understanding of the dynamics of complex microbial communities inhabiting the human body. Bacteriophages have a substantial capacity to alter prokaryote communities, and because perturbations in prokaryote communities contribute to disease processes, analysis of bacteriophages may provide important clues as to the development of both periodontitis and dental caries in our human subjects. The implications for disease development render this research relevant to public health, and may provide a new focal point for the development of tests for the diagnosis and prediction of disease associated with human microbial communities.