Vibrio fischeri as a Model for Bacterial Colonization

项目摘要/摘要-拟议研究方案的长期目标是提供对 在关键时期，每一代人与他们的微生物群之间发生的复杂对话 出生后的时期。最近的研究表明，这些早期疾病的结果之间存在很强的相关性 活动和终身健康。然而，定植组织的不可获得性和生物的高度多样性 微生物群使得对人类组织早期定植的深入研究具有极大的挑战性。当面对时 对于如此复杂的现象，生物学家经常求助于更简单的模型系统来提供对 进化上保守的特征，揭示了基本原理。为了破译细胞和分子 细菌与粘膜上皮细胞顶面结合的机制 拟议的计划利用了鱼弧菌与其鱿鱼宿主之间的二元共生关系， Euprymna Scolope。这种相对简单的联系已经被研究了20多年，作为 革兰氏阴性菌对粘膜的慢性定植。和人类一样，鱿鱼-弧菌协会 每一代都会重新开始，需要对其他环境细菌进行筛选，从而产生持久的 联合仅限于共同进化的伙伴。在这个系统中，发生了共生启动的过程。 在几分钟到几个小时内跨越~100微米。可以使用共聚焦显微镜直接对其整体成像， 这提供了难得的机会来定义具有高时间和空间分辨率的倒易分子 以及生化对话，从而建立一种特定的、终身有益的共生关系。这 该项目汇集了两个合作者，每个人都拥有其中一个共生伙伴的生物学专业知识， 并将新技术引入到宿主-微生物相互作用的研究中，包括：纳米二次离子 质谱学(NanoSIMS)，允许精确跟踪共生体分子进入宿主组织； 杂交-链式反应荧光原位杂交(HCR-FISH)，使可视化 宿主细胞和共生体细胞中罕见的转录本；纳米串，一种同时分析数十个基因的新技术 到数百个靶向转录本；以及产生强大转录文库的高效RNAseq 从小到10 ng的总RNA(约105个细菌)。要解决的具体目标是：(1)审查如何 共生细菌菌株的变异影响共生的开始和持续；(2)外源细菌的特性 膜泡(OMV)含量，它们向宿主细胞的转运，以及宿主对OMV货物的反应；以及，(3) 对弧菌毒力决定因素在非致病关联中的作用的研究。一个 对人类微生物组的认识还处于初级阶段，这一前沿领域目前正处于 构建范例。在这方面，正如鱿鱼-弧菌系统在过去所做的那样，目前 研究将阐明控制有益和致病的基本原则。 联想。

Project Summary/Abstract - 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: Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which allows precision tracking of symbiont molecules into host tissues; Hybridization-Chain-Reaction Fluorescent In Situ Hybridization (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). Specific aims to be addressed are: (1) the examination of how symbiotic bacterial strain variation affects symbiosis onset and persistence; (2) the characterization of outer membrane vesicle (OMV) contents, their trafficking into host cells, and host responses to OMV cargo; and, (3) the investigation of the roles of vibrio virulence determinants in a non-pathogenic association. 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.