Vibrio fischeri as a Model for Bacterial Colonization

项目摘要/摘要-拟议研究计划的长期目标是提供洞察力， 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，这些早期的结果之间有很强的相关性。 事件和终身健康。然而，难以获得定植组织和高度多样性的 微生物群的变化使得对人体组织早期定殖的深入研究极具挑战性。面对 面对如此复杂的现象，生物学家们常常求助于更简单的模型系统， 进化上保守的特征和揭示基本原则。去破译细胞和分子 细菌与粘膜上皮顶端表面结合的启动机制， 所提出的方案利用了细菌费氏弧菌和其鱿鱼宿主之间的二元共生关系， 黄翅真樱这种相对简单的关联已经被研究了二十多年，作为一种模型， 革兰氏阴性菌在粘膜的慢性定植。就像在人类身上一样，乌贼弧菌 每一代都重新开始，需要对其他环境细菌进行“筛选”，导致持续的 仅限于共同进化的伙伴。在这个系统中，共生启动的过程发生 在几分钟到几小时的时间内跨越约100微米。可以使用共聚焦显微镜直接对其整体成像， 它提供了难得的机会，以高的时间和空间分辨率，确定相互分子 和生物化学的对话，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，每个人都具有共生伙伴之一的生物学专业知识， 介绍了研究宿主-微生物相互作用的新技术，包括：纳米级二次离子 质谱法（NanoSIMS），可以精确追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），使可视化的 宿主和共生体细胞中的罕见转录本; NanoString，一种同时分析数十种 到数百个靶向转录本;和高效RNAseq，它产生强大的转录文库 从低至10 ng的总RNA（约105个细菌）。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久性;（2）外 膜囊泡（OMV）内容物，它们向宿主细胞的运输，以及宿主对OMV货物的反应;以及（3） 弧菌毒力决定因子在非致病性关联中作用的研究。一个 对人类微生物组的了解尚处于起步阶段，这一前沿领域目前正处于 构建范例。在这种情况下，正如鱿鱼弧菌系统在过去所做的那样，目前的结果是 这项研究将阐明控制有益和致病疾病发生的基本原则， 协会.

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.