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深入了解， 在出生后的关键时期，人类和他们的微生物群之间的每一代都会发生。 最近的研究表明，这些早期事件的结果与终身疾病之间存在很强的相关性。 健康然而，定植组织的不可接近性和微生物群的高度多样性使得 深入研究人类组织的早期定殖极具挑战性。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， 启动细菌协会与顶端表面的粘膜上皮，提出的计划利用 费氏弧菌和它的乌贼宿主--真乌贼之间的二元共生关系。这 相对简单的关联作为长期殖民的模型已经研究了二十多年 革兰氏阴性菌对粘膜的影响。就像在人类中一样，鱿鱼-弧菌的关系在每一次 产生，需要“筛选”其他环境细菌，导致持久的关联 仅限于共同进化的伙伴。在这个系统中，共生开始的过程发生在约100 几分钟到几个小时。它可以直接成像在其整体使用共聚焦显微镜， 提供了难得的机会，以高的时间和空间分辨率，确定相互的分子和 生物化学对话，导致建立一个特定的，终身有益的共生。这 该项目汇集了两个合作者，每个人都有一个共生生物学的专业知识， 合作伙伴，并介绍了新技术的研究宿主微生物的相互作用，包括：HCR-FISH， 这使得宿主和共生体细胞中罕见的转录本可视化; NanoString，一种用于 同时分析数十到数百个靶向转录本;以及高效RNAseq， 从低至10 ng的总RNA（约105个细菌）产生稳健的转录文库。的理解 人类微生物组的研究尚处于起步阶段，这一前沿领域目前正处于建设阶段。 范例在此背景下，正如鱿鱼弧菌系统过去所做的那样，当前研究的结果 将阐明控制有益和致病的发病的基本原则， 协会. 相关性（见说明）：在人类和其他动物的定植过程中，无论是正常的 微生物或微生物病原体，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 无论是一个健康的协会将被培养，一个功能失调的社区将发展，还是一个坦率的 发病机制将随之而来。我们将使用创新的成像，微生物遗传学和基因组学/转录组学 方法来发现这一关键时期的分子机制，定义一般的 控制宿主细胞和微生物之间相互作用的原理。

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.