Elucidating Molecular-Level Roles of Essential Metals in Gut Bacteria with New Fluorescent Protein-Based Metal Ion Sensors

项目摘要 饮食中金属营养素的变化，包括锌和铁，影响微生物群的组成 并与感染易感性增加和胃肠道疾病相关，但其分子机制 这些影响的根本原因在很大程度上仍不清楚。这种知识的缺乏严重限制了我们预测 饮食或宿主金属状态将影响胃肠道疾病或感染的治疗。我们的长期目标是 阐明必需金属如何影响人类肠道微生物群的分子机制。的 拟议工作的总体目标是确定必需金属如何影响生长和交流 在乳杆菌属物种的益生菌群落中。我们的研究策略是1）开发和应用 基于蛋白质的荧光传感器，不依赖于氧气和2）揭示分子机制， 哪些金属离子影响肠道微生物群的稳态。氧不敏感的蛋白质荧光传感器将 用于含有乳酸杆菌的活厌氧培养物中，以研究金属吸收和金属离子水平如何变化 随着时间纯的、多物种的和体外肠道模型培养物将用于评估金属离子体内平衡如何 随着细菌种类的增加和复杂性的增加而变化。除了直接探测和跟踪 我们正在进行系统的研究，以测量如何使用荧光传感器在文化中的必需金属， 必需金属的变化影响乳酸菌生理学和细胞间通讯（群体感应）。在这里， 我们正在研究乳酸杆菌储存过量金属离子的能力，并旨在确定 生长培养物中受不同金属水平影响的基因。我们也在测量不同的金属水平如何影响 大量的乳酸菌群体感应信号分子。这项研究计划得到加强， 与微生物学，微生物组和先进荧光显微镜专家的合作。研究 是重要的，因为它将提供膳食金属如何影响肠道微生物群组成的机制见解 和功能这一见解是重要的，因为它将有助于预测金属基饮食的影响， 这些干预措施可能会确定新的目标，以减轻这些影响。此外，它将提供一个 益生菌饮食干预的知识基础，以对抗胃肠道疾病，并可能识别 新的药物靶点这项研究是创新的，因为它代表了对当前工作的实质性偏离 通过转移焦点，揭示肠道细菌中金属离子的分子水平机制和作用， 微生物群的组成和功能。通过研究乳杆菌属，我们利用了公认的 遗传方法，而专注于丰富的有机体在小肠，其中大多数金属营养素 发生了吸收。此外，乳酸杆菌作为益生菌被广泛接受，但仍有许多有待了解的问题。 其有益的作用机制。开发新的基于蛋白质的金属传感器，以克服氧- 目前基于蛋白质的传感器的依赖性将允许检测细菌金属摄取和暴露， 培养和在生理（厌氧）条件下的体外肠道模型中。

PROJECT SUMMARY Dietary changes in metal nutrients, including zinc and iron, influence the composition of the microbiota and correlate with increased infection susceptibility and gastrointestinal diseases, but the molecular mechanisms underlying these effects remain largely unknown. This lack of knowledge severely limits our ability to predict how diet or host metal status will impact treatment of gastrointestinal diseases or infection. Our long-term goal is to elucidate the molecular mechanisms governing how essential metals affect the human gut microbiota. The overall objective of the proposed work is to determine how essential metals affect growth and communication within probiotic bacterial communities of Lactobacillus species. Our research strategy is 1) to develop and apply protein-based fluorescent sensors that do not rely on oxygen and 2) to uncover molecular mechanisms through which metal ions affect gut microbiota homeostasis. Oxygen-insensitive protein-based fluorescent sensors will be used in live anaerobic cultures containing Lactobacillus to study metal uptake and how metal ion levels vary over time. Pure, multispecies, and in vitro gut model cultures will be used to evaluate how metal ion homeostasis varies with additional bacterial species and increasing complexity. Beyond direct detection and tracking of essential metals in culture with fluorescent sensors, we are carrying out systematic studies to measure how changes in essential metals affect Lactobacillus physiology and cell-cell communication (quorum sensing). Here, we are investigating the capacity of Lactobacillus species to store excess metal ions and aiming to identify the genes affected by varied metal levels in growth cultures. We are also measuring how varied metal levels affect the abundance of Lactobacillus quorum sensing signaling molecules. This research program is enhanced by collaborations with experts in microbiology, microbiome, and advanced fluorescence microscopy. The research is significant because it will provide mechanistic insight to how dietary metals affect gut microbiota composition and function. This insight is important because it will be useful for predicting the effects of metal-based dietary interventions and could potentially identify new targets to mitigate these effects. Furthermore, it will provide a knowledge basis for probiotic dietary interventions to combat gastrointestinal diseases and potentially identify new drug targets. The research is innovative because it represents a substantive departure from current work by shifting focus to uncover molecular-level mechanisms and roles for metal ions in gut bacteria that affect microbiota composition and function. By studying the Lactobacillus genus, we take advantage of well-established genetic approaches while focusing on an abundant organism in the small intestine, where most metal nutrient uptake occurs. Furthermore, Lactobacillus are well accepted as probiotics, but much remains to be learned about their beneficial mechanisms of action. Developing new protein-based metal sensors to overcome the oxygen- dependency of current protein-based sensors will allow detection of bacterial metal uptake and exposure in culture and in in vitro gut models under physiological (anaerobic) conditions.