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

项目总结 饮食中金属营养素的变化，包括锌和铁，会影响微生物区系的组成 并与感染易感性增加和胃肠道疾病有关，但分子机制 这些影响的潜在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 饮食或宿主金属状况将影响胃肠道疾病或感染的治疗。我们的长期目标是 阐明控制必需金属如何影响人体肠道微生物区系的分子机制。这个 拟议工作的总体目标是确定必需金属如何影响增长和交流。 在乳杆菌种的益生菌群落内。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，不依赖氧气和2)通过 哪些金属离子影响肠道微生物区系的动态平衡。氧不敏感的蛋白质荧光传感器将 在含有乳酸菌的厌氧活培养中用于研究金属的吸收和金属离子水平的变化 随着时间的推移。纯、多物种和体外肠道模型培养将被用来评估金属离子的动态平衡 随着细菌种类的增加和复杂性的增加而变化。除了直接检测和跟踪 利用荧光传感器，我们正在进行系统的研究，以测量 必需金属的变化会影响乳杆菌的生理和细胞间的通讯(群体感应)。这里, 我们正在调查乳杆菌物种储存多余金属离子的能力，并旨在确定 在生长培养中受不同金属水平影响的基因。我们还在测量不同的金属水平如何影响 群体乳杆菌感应信号分子的丰度。这项研究计划通过以下方式得到加强 与微生物学、微生物组和高级荧光显微镜方面的专家合作。这项研究 具有重要意义，因为它将提供关于膳食金属如何影响肠道微生物区系组成的机械洞察力 和功能。这一见解很重要，因为它将有助于预测以金属为基础的饮食的影响 此外，还需要采取干预措施，并有可能确定新的目标来减轻这些影响。此外，它还将提供一个 用于对抗胃肠道疾病的益生菌饮食干预的知识基础 新的药物靶点。这项研究具有创新性，因为它代表了对当前工作的实质性偏离 通过转移焦点来揭示影响肠道细菌中金属离子的分子水平的机制和作用 微生物区系组成和功能。通过研究乳杆菌属，我们利用了历史悠久的 遗传方法，同时专注于小肠中丰富的有机体，在那里大多数金属营养 就会发生摄取。此外，乳杆菌作为益生菌被广泛接受，但仍有许多事情需要了解 它们有益的作用机制。开发基于蛋白质的新型金属传感器，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对金属的摄取和暴露 生理(厌氧)条件下的培养和体外肠道模型。

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.