Vibrio fischeri as a Model for Bacterial Colonization

拟议研究计划的长期目标是提供对复杂对话的洞察， 在出生后的关键时期，每一代人与其微生物群之间都会发生这种情况。 最近的研究表明，这些早期事件的结果与终生之间存在很强的相关性 健康。然而，定植组织的不可获得性和微生物区系的高度多样性导致了 深入研究人类组织的早期定植极具挑战性。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， 发起细菌与粘膜上皮顶端表面的联系，拟议的计划利用 鱼弧菌与其鱿鱼宿主真乌贼的二元共生。这 20多年来，人们一直在研究相对简单的关联，作为慢性殖民的模型 革兰氏阴性杆菌对粘膜的致病菌。与人类一样，鱿鱼与弧菌的联系每次都会重新开始。 世代，需要对其他环境细菌进行‘筛选’，从而产生持久的联想 仅限于共同进化的伙伴。在这个系统中，共生的启动过程发生在~100 几分钟到几个小时的微米。可以使用共聚焦显微镜直接对其整体进行成像， 提供了难得的机会来定义具有高时间和空间分辨率的相互作用的分子和 建立一种特定的、终生有益的共生关系的生化对话。这 该项目汇集了两个合作者，每个人都拥有其中一种共生生物的生物学专业知识 并将新技术引入宿主-微生物相互作用的研究，包括：HCR-FISH， 这使得宿主细胞和共生体细胞中罕见的转录物可视化成为可能；纳米串是一种用于 同时分析数十到数百个目标转录本；以及高效RNAseq，它 从低至10 ng的总RNA(~105个细菌)产生强大的转录文库。一种理解 人类微生物群的研究还处于初级阶段，这一前沿领域目前正处于构建阶段 范例。在这一背景下，正如鱿鱼-弧菌系统过去所做的那样，目前的研究结果 将阐明控制有益和致病的基本原则 联想。 相关性(见说明)：在人类和其他动物的殖民期间，由正常的 对于微生物区系或微生物病原体，最初的几个小时是至关重要的。由此产生的相互作用决定了 无论是培养一个健康的协会，一个功能失调的社区，还是一个坦率的 发病机制将随之而来。我们将使用创新成像、微生物遗传学和基因组学/转录学 发现这一关键时期背后的分子机制的方法，定义了一般的 管理宿主细胞和它们的微生物之间相互作用的原则。

The long-term goals of the proposed research program are to provide insight into the complex dialogue that occurs each generation between humans and their microbiota during the critical period following birth. Recent research has indicated a strong correlation between outcomes of these early events and life-long health. However, the inaccessibility of colonized tissues and high diversity of the microbiota renders an in- depth study of early colonization of human tissues 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 initiation of bacterial associations with apical surfaces of mucosal epithelia, the proposed program exploits the binary symbiosis between the bacterium Vibrio fischeri and its squid host, Euprymna scolopes. This relatively simple association has been studied for over two decades as a model for the chronic colonization of mucosa by Gram-negative bacteria. As in humans, the squid-vibrio association begins anew each generation, requiring a ‘winnowing’ of other environmental bacteria that results in persistent association restricted to the coevolved partners. In this system, the process of symbiosis initiation occurs across ~100 microns over minutes to hours. It can be directly imaged in its entirety using confocal microscopy, which offers the rare opportunity to define, with high temporal and spatial resolution, the reciprocal molecular and biochemical dialogue that results in the establishment of a specific, life-long beneficial symbiosis. This project brings together two collaborators, each with expertise in the biology of one of the symbiotic partners, and introduces new technology to the study of host-microbe interactions, including: HCR-FISH, which enables visualization of rare transcripts in host and symbiont cells; NanoString, a new technology 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 (~105 bacteria). 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 governing the onset of both beneficial and pathogenic associations. RELEVANCE (See instructions): During the colonization of humans and other animals, by either the normal microbiota or microbial pathogens, the first few hours are critical. The resulting interplay determines whether a healthy association will be fostered, a dysfunctional community will develop, or a frank pathogenesis will ensue. We will use innovative imaging, microbial genetics, and genomics/transcriptomics approaches to discover the molecular mechanisms underlying this crucial period, defining the general principles that govern the reciprocal interactions between host cells and their microbes.