Vibrio colonization determinants in symbiosis

项目摘要/摘要 拟议研究计划的长期目标是提供对复杂对话的洞察， 在人类和他们正常的有益微生物群之间进化。最近的研究表明， 成功维护这一对话与终身健康之间的关系。然而，这种不可访问性 哺乳动物的定植组织及其微生物群落的高度多样性为深入研究 人类肠道的持久殖民轨迹极具挑战性。当面对如此复杂的情况时 生物学家经常求助于更简单的模型系统来洞察进化上保守的 特征和揭示基本原理。为了破译细胞和分子机制， 细菌与粘膜上皮细胞顶端表面的慢性关联，拟议的计划利用了 鱼弧菌与其鱿鱼寄主Euprymna scolope之间特殊的二元共生。这 离散的、实验上容易处理的关联作为一种模型已经研究了近30年 革兰氏阴性菌对粘膜的长期定植。和人类一样，鱿鱼-弧菌协会 每一代人都会重新开始，并促进双方的持续健康。这种共生关系可以 用具有明确突变的费氏弧菌菌株启动，宿主可以繁殖并在其整个 生活。这种联系可以用共聚焦显微镜直接成像，这提供了难得的机会 以高时间和空间分辨率定义一生中相互作用的分子和基因对话 受益协会。该项目结合了两个合作绩效指标的专业知识，每个项目都具有 其中一个共生伙伴的生物学，以及三个合作者的额外分析专业知识。一起， 他们介绍了研究宿主-微生物相互作用的新方法和新技术，包括：相互作用 共生对双方影响的表观基因组分析；杂交-连锁反应，荧光素 原位杂交(HCR-FISH)，它使宿主细胞和共生体细胞中罕见的转录本得以可视化 定植组织；纳米串技术，一种同时分析数十到数百个 靶向转录本；以及高效的RNAseq，它从尽可能少的时间产生强大的转录文库 总RNA为10 ng(约100,000个细菌)。要解决的具体目标是：(I)确定如何每天 代谢节律在宿主和其微生物群之间协调；(Ii)发现共生体如何 维持一个有节奏的生长和持久性周期；以及，(Iii)确定表观遗传修饰在 发展和维持有益的共生关系。对人类微生物群的了解在 它还处于起步阶段，这一前沿领域目前正处于构建范式的阶段。在此上下文中，作为 鱿鱼-弧菌系统在过去有过，目前的研究结果将阐明基本原理 管理有益微生物和致病微生物的持续殖民。

Project Summary/Abstract The long-term goals of the proposed research program are to provide insight into the complex dialogue that has evolved between humans and their normal beneficial microbiota. Recent research has indicated a strong correlation between the successful maintenance of this dialog and life-long health. However, the inaccessibility of colonized tissues in mammals and the high diversity of their microbiomes render an in-depth study of persistent colonization of the human gut track extremely challenging. When faced with such complex phenomena, biologists often turn to simpler model systems to provide insights into evolutionarily conserved features and reveal basic principles. To decipher the cellular and molecular mechanisms underlying the chronic association of bacteria with apical surfaces of mucosal epithelia, the proposed program exploits the specific, binary symbiosis between the bacterium Vibrio fischeri and its squid host, Euprymna scolopes. This discrete, experimentally tractable, association has been studied for almost three decades as a model for the long-term colonization of mucosa by Gram-negative bacteria. As in humans, the squid-vibrio association begins anew each generation, and fosters the continuing health of both partners. The symbiosis can be initiated with V. fischeri strains with defined mutations, and the host can be bred and maintained for its entire life. The association can be directly imaged using confocal microscopy, which offers the rare opportunity to define, with high temporal and spatial resolution, the reciprocal molecular and genetic dialogue in a life-long beneficial association. This project combines the expertise of the two co-PIs, each with experience in the biology of one of the symbiotic partners, with additional analytical expertise of three collaborators. Together, they introduce new approaches and technology to the study of host-microbe interactions, including: reciprocal epigenomic analyses of the effect of symbiosis on both partners; hybridization-chain-reaction, fluorescent in situ hybridization (HCR-FISH), which enables visualization of rare transcripts in both host and symbiont cells in colonized tissues; NanoString Technology, a new method for simultaneous analysis of dozens to hundreds of targeted transcripts; and, high-efficiency RNAseq, which produces robust transcriptional libraries from as little as 10 ng total RNA (~100,000 bacteria). Specific aims to be addressed are: (i) determining how a daily metabolic rhythm is coordinated between a host and its microbiome; (ii) discovering how the symbionts maintain a rhythmic cycle of growth and persistence; and, (iii) defining the role of epigenetic modifications in the development and maintenance of a beneficial symbiosis. An understanding of the human microbiome is in its infancy, and this frontier field is currently at the stage of building paradigms. Within this context, as the squid-vibrio system has in the past, the results of the current study will shed light upon fundamental principles that govern persistent colonization by both beneficial and pathogenic microbes.