High-Resolution Mapping of Colonization Factors in a Model Microbiome

模型微生物组中定植因子的高分辨率绘图

• 批准号: 10309044
• 负责人: Hector Luis Burgos
• 金额: $ 6.86万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10309044
• 关键词: Resolution; Mapping; Colonization; Factors; Model

PROJECT SUMMARY Animals form stable and reproducible associations with specific microbes that are essential for health. However, the molecular mechanisms that result in the selection and retention of the appropriate beneficial symbionts are understudied. Here we use the beneficial symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri as a model system to study the molecular dialogue that occurs at the microbe-host interface to ensure specificity in the interaction. Using a global transposon insertion sequencing approach (INSeq/Tn-seq) our lab previously identified 344 putative novel colonization factors. Pilot validation of a subset of these candidates showed that approximately half are true colonization factors. This approach identified factors ranging across various molecular functions, including amino acid metabolism, motility, biofilm formation, and signal transduction, as well as a number of so-called hypothetical proteins. Various copper exporters were also identified as validated colonization factors, suggesting that copper is playing a role at the microbe-host interface in this marine system. Further study of the identified factors required clean deletions, but we were limited in obtaining these due to the complexity of the random libraries generated via transposon mutagenesis and the time-consuming process of plasmid-based allelic exchange. To address these obstacles, I developed an updated mutagenesis approach to quickly generate barcoded in-frame gene deletions in V. fischeri. Barcode sequencing (BarSeq) can then be used to track population dynamics and perform high- throughput competition experiments with defined populations of deletion mutants. The overall goal of this proposal is to characterize the molecular pathways and functions that are responsible for the establishment of the specific and reproducible beneficial symbiosis between V. fischeri and its squid host. To this end, I used the method I developed to generate 48 barcoded deletions, including several copper exporters. In Aim 1, I will continue using this approach to generate barcoded deletions of all 344 novel putative colonization factors. In addition, barcoded mutants will be used in Aim 1 to perform high-throughput competitive squid colonization experiments using BarSeq to track population dynamics and determine colonization efficiency for each mutant strain. This approach will be used to define stages of host colonization with high resolution. Preliminary experiments have validated additional copper export mutants as true colonization factors. In Aim 2, I will use a combination of BarSeq and traditional biochemical and molecular biology techniques to characterize the role that copper has during squid colonization by V. fischeri. Completion of these aims will generate a valuable deletion library resource that will be used to characterize the molecular factors that determine specificity during establishment of the beneficial vibrio-squid symbiosis. In addition, determining the role of copper during squid colonization will illuminate mechanisms that act during establishment of a beneficial symbiosis that may also be shared with pathogens.

项目概要 动物与对健康至关重要的特定微生物形成稳定且可重复的关联。然而， 导致选择和保留适当的有益共生体的分子机制是 待研究。在这里，我们利用夏威夷短尾鱿鱼 Euprymna scolopes 之间的有益共生关系 以及生物发光细菌费氏弧菌作为模型系统来研究发生的分子对话 在微生物-宿主接口处，以确保相互作用的特异性。使用全局转座子插入 测序方法 (INSeq/Tn-seq) 我们的实验室之前鉴定了 344 个假定的新型定植因子。飞行员 对这些候选者的子集的验证表明，大约一半是真正的定植因子。这 方法确定了各种分子功能的因素，包括氨基酸代谢、运动性、 生物膜形成、信号转导以及许多所谓的假设蛋白质。各种铜 出口商也被确定为有效的殖民因素，这表明铜在 该海洋系统中的微生物-宿主界面。对已确定因素的进一步研究需要彻底删除，但是 由于通过转座子生成的随机库的复杂性，我们获得这些数据受到限制 诱变和基于质粒的等位基因交换的耗时过程。为了解决这些障碍， 我开发了一种更新的诱变方法，可以在 V 中快速生成带有条形码的框内基因删除。 费舍里。然后，条形码测序 (BarSeq) 可用于跟踪种群动态并执行高通量测序。 与确定的缺失突变体群体进行吞吐量竞争实验。本次活动的总体目标 提议是描述负责的分子途径和功能 费氏弧菌与其鱿鱼之间建立特异性且可重复的有益共生关系 主持人。为此，我使用我开发的方法生成了 48 个条形码删除，其中包括几个铜 出口商。在目标 1 中，我将继续使用这种方法来生成所有 344 个小说假定的条形码删除 定植因素。此外，带有条形码的突变体将用于Aim 1中以进行高通量竞争 使用 BarSeq 进行鱿鱼定植实验来跟踪种群动态并确定定植效率 对于每个突变株。该方法将用于以高分辨率定义宿主定植的阶段。 初步实验已验证额外的铜输出突变体是真正的定植因子。在目标 2 中， 我将结合使用 BarSeq 和传统的生化和分子生物学技术来表征 铜在费氏弧菌定植鱿鱼过程中的作用。完成这些目标将产生 宝贵的缺失文库资源，将用于表征决定特异性的分子因素 在建立有益的弧菌-鱿鱼共生关系期间。此外，确定铜在过程中的作用 鱿鱼定植将阐明在建立有益共生关系过程中发挥作用的机制，该共生关系可能 也与病原体共享。