Chemotactic Cues in the Symbiosis Between Vibrio fischeri and Euprymna scolopes

描述（由申请人提供）：宿主-微生物相互作用，包括有益的和致病的，是一个快速发展的研究领域，对理解正常微生物群以及感染对其的干扰具有重要意义。生物发光细菌弧菌fischeri和夏威夷短尾鱿鱼，Euprymna candiopes之间的单种共生，为我们提供了一个理想的模型，以研究共生开始的最早期事件。这项提案将集中在一个关键的细菌信号通路，趋化性，它允许细菌识别和响应其环境的变化。在有益的宿主-微生物关联的背景下表征细菌趋化性将提供微生物如何与宿主组织呈现的复杂环境相互作用和响应的更完整的画面，同时从环境海水迁移到宿主生物体中。 本研究的总体目标是表征在细菌趋化机制中作为配体受体的甲基接受趋化蛋白（MCP），以及这些受体的调节。迄今为止，在E.杆菌然而，E.大肠杆菌只使用其中的五种受体，而大多数大肠杆菌都有更多的受体。预计费氏弧菌编码43种MCP，并且已知它们的表达与宿主的定殖相关。E.费氏弧菌 已经证明，在几个不同的阶段中发生，并且涉及细菌在从浮游细菌移动到动物的内部发光器官时通过几个环境和组织的迁移。本研究将进一步阐明V. fischeri，其中哪些参与了共生的开始。第一个目标是解决受体-配体相互作用，并将使用已建立的技术，如软琼脂运动试验和毛细管迁移试验，以及基于FRET的试验，允许在无受体背景菌株中研究特异性配体-蛋白相互作用。杆菌第二个目标将研究这些MCP的调节，并将利用RNAseq和荧光报告方法的转录谱分析，在设计用于确定定殖的不同阶段是否涉及特定受体的实验中。在本项目过程中使用和开发的工具也将适用于其他研究，无论是在Ruby实验室还是在其他研究宿主-微生物相互作用的实验室。该项目还将在几个领域为博士后研究员提供重要的培训，包括先进的显微镜和转录分析技术。

DESCRIPTION (provided by applicant): Host-microbe interactions, both beneficial and pathogenic, are a rapidly developing area of research with important implications for understanding the normal microbiota, as well as its disturbance by infection. The monospecific symbiosis between the bioluminescent bacteria Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, provides us with an ideal model with which to study the earliest events of symbiotic initiation. This proposal will focus on a key bacterial signaling pathway, chemotaxis, which allows a bacterium to recognize and respond to changes in its environment. Characterization of bacterial chemotaxis in the context of a beneficial host-microbe association will provide a more complete picture of how microbes interact with and respond to the complex environments presented by host tissues, while migrating from the ambient seawater into a host organism. The overall goals of this research are to characterize the methyl-accepting chemotaxis proteins (MCPs) which act as the ligand receptors in the bacterial chemotaxis machinery, as well as the regulation of these receptors. MCPs have been well studied to date in E. coli. However, E. coli only uses a set of five of these receptors, while the majority of bacteri are known to have many more. V. fischeri is predicted to encode 43 MCPs, and their expression is known to be relevant to colonization of the host. The colonization of E. scolopes by V. fischeri has been shown to occur in several distinct stages, and involves the bacterium migrating through several environments and tissues as it moves from the bacterioplankton to the interior light organ of the animal. This research will further clarify the specific ligands recognized by V. fischeri, and which of these are involved during the initiation of symbiosis. The first aim will address the receptor-ligand interactions, and will be pursued using established techniques such as soft-agar motility assays and capillary-tube migration assays, as well as a FRET-based assay that allows investigation of specific ligand-protein interactions in a receptorless- background strain of E. coli. The second aim will investigate the regulation of these MCPs, and will utilize both transcriptional profiling by RNAseq and fluorescent reporter methods, in experiments designed to determine whether distinct stages of the colonization involve specific receptors. The tools used and developed during the course of this project will also be applicable to other studies, both in the Ruby lab and in other labs studying host-microbe interactions. This project will also provide significant training to the post-doctoral fellow in several areas, includng advanced microscopy and transcriptional profiling techniques.