Vibrio colonization determinants in symbiosis

项目总结/摘要 拟议研究计划的长期目标是提供对复杂对话的深入了解， 是在人类和正常的有益微生物群之间进化的。最近的研究表明， 此对话框的成功维护与终身健康之间的相关性。然而， 在哺乳动物的殖民组织和他们的微生物的高度多样性，使深入研究 人类肠道的持续殖民极具挑战性。当面对如此复杂的 生物学家经常转向更简单的模型系统，以提供对进化保守的见解。 特点，揭示基本原理。为了破译细胞和分子机制， 慢性协会的细菌与顶端表面的粘膜上皮，提出的计划利用 在细菌费氏弧菌和它的鱿鱼宿主，Euprymna sputteropes之间的特殊的二元共生。这 离散的，实验上易于处理的，协会已经研究了近三十年，作为一个模型， 革兰氏阴性菌在粘膜的长期定植。就像在人类身上一样，乌贼弧菌 每一代都重新开始，并促进双方的持续健康。共生关系可以是 起始于具有确定突变的费氏弧菌菌株，并且宿主可以繁殖并维持其整个 生活该协会可以直接成像使用共聚焦显微镜，这提供了难得的机会， 以高的时间和空间分辨率，定义一生中相互的分子和遗传对话， 有益的联系。该项目结合了两名共同首席执行官的专业知识，每个人都有经验， 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。在一起， 它们为宿主-微生物相互作用的研究引入了新的方法和技术，包括： 共生对双方影响的表观基因组分析; 原位杂交（HCR-FISH），它使宿主和共生体细胞中的罕见转录本可视化， NanoString技术，一种同时分析数十到数百个 靶向转录物;以及高效RNAseq，它可以从尽可能少的 作为10 ng总RNA（~ 100，000个细菌）。要解决的具体目标是：（一）确定如何每天 代谢节律在宿主和其微生物组之间协调;（ii）发现共生体如何 维持生长和持久性的有节奏的循环;以及（iii）确定表观遗传修饰在以下方面的作用： 发展和维持有益的共生关系。对人类微生物组的了解是在 这一前沿领域目前正处于建立范式的阶段。在这方面，作为 鱿鱼弧菌系统在过去，目前的研究结果将阐明基本原则 控制着有益微生物和致病微生物的持续定植。

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.