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Tracking, elucidation and modulation of xenometal homeostasis in bacteria

细菌异种金属稳态的追踪、阐明和调节

基本信息

批准号：
10651734
负责人：
Eszter Boros
金额：
$ 37.88万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2026-06-30
项目状态：
未结题

项目摘要

Bacterial virulence is closely associated with nutrient acquisition, which is essential for growth and proliferation of pathogens. Metal ions constitute essential nutrients, and the regulation of bacterial metal ion homeostasis within the host environment plays a pivotal role; however, unbound essential metal ions exhibit low bioavailability. For instance, the low solubility of Fe(OH)3 (Ksp = 6.3 x 10-38) at pH 7.4 would result in an insufficient quantity of iron for bacteria to grow, thus bacteria rely on targeting the hosts’ labile iron reserves through synthesis of endogenous, hydrophilic metallophores that are internalized using ATP-dependent bacterial transmembrane shuttles. These metallophores also retain affinity for non-essential xenometal ions with identical charge, comparable ionic radius and chemical hardness to the essential metal ion. For instance, trivalent metal ions with similar ionic radius to high spin Fe3+ (0.78 Å), such as Ga3+ (0.76 Å), Sc3+ (0.87 Å) and In3+ (0.93 Å) are transported to the bacterial peri- and cytoplasm when coordinated by bacterial iron-metallophores such as enterobactin or desferioxamine. These xenometals cannot be utilized for desired biological functions; recent strategies to utilize bacterial metal homeostasis pathways to deliver therapeutics has resulted in renewed interest in xenometals as alternative antibiotics. In bacteria, iron’s cytoplasmic fate and influence on gene and protein regulation is well-understood; however, xenometal homeostasis and utilization, especially in light of differential pH-dependent speciation behavior, remains rudimentary. To this end, we seek to investigate the following questions: (1) Are M3+-metallophore complexes efficiently recognized and transported across bacterial membranes? Size, hardness and Lewis acidity of metal ions influence their coordination complex structure. Substantial divergence from the parent Fe3+ complex results in diminished transport efficiency. We will study xenometal complex speciation under physiological conditions and employ a photoreactive tagging strategy to identify transmembrane shuttle protein interaction. (2) (How) Does M3+ release from metallophores proceed in absence of accessible redox events? Fe3+ is released by reduction to Fe2+ and enzymatic degradation of the metallophore induced by Fe2+-dependent proteins. The xenometals of interest, Ga3+, Sc3+ and In3+, do not have accessible redox events under physiological conditions. We will employ a radiochemical labeling strategy to track their metallophore-mediated uptake and identify metabolites. (3) What is the fate of M3+ xenometals in the cytoplasm and their influence on protein expression? The fate of non-redox active xenometals, once they reach the bacterial cytoplasm, including their effect on the bacterial protein expression is not well understood but hold the key to their growth inhibitory activity. We will combine radiochemical tagging strategies with mass spectrometry isolate and identify xenometal-target proteins. We will assess and quantitate the change in bacterial metabolites following exposure to different xenometal- metallophore complexes, which will inform on altered bacterial metabolism.

细菌的毒力与营养物质的获取密切相关，营养物质的获取对细菌的生长和繁殖至关重要。病原体的数量。金属离子构成人体必需的营养物质，并调节细菌金属离子的动态平衡在宿主环境中起着关键作用；然而，非结合的必需金属离子表现出较低的生物利用度。例如，在pH 7.4时，Fe(OH)3(KSP=6.3×10-38)的低溶解度将导致细菌生长所需的铁，因此细菌依赖于通过合成利用依赖于三磷酸腺苷的细菌跨膜内化的内源亲水性金属载体航天飞机。这些金属载体还保持了对具有相同电荷的非必需的金属离子的亲和力，离子半径和化学硬度与基本金属离子相当。例如，三价金属离子与与高自旋Fe3+(0.78？)，如Ga3+(0.76？)，Sc3+(0.87？)和In3+(0.93？)的离子半径相似当被细菌铁金属载体协调时，运输到细菌的周缘和细胞质肠杆菌素或去铁胺。这些异族金属不能用于预期的生物功能；最近利用细菌金属动态平衡途径传递治疗药物的策略重新引起了人们的兴趣在异族金属中作为替代抗生素。在细菌中，铁在细胞质中的命运和对基因和蛋白质调节的影响是众所周知的；然而，异族金属的动态平衡和利用，特别是考虑到不同的依赖于pH的形态行为，仍处于初级阶段。为此，我们试图研究以下问题：(1)M3+-金属基团复合体能有效识别和跨细菌膜传输吗？尺寸、硬度和刘易斯酸度金属离子的含量影响它们的配位络合物结构。与母体Fe3+络合物的显著差异导致运输效率降低。我们将在生理条件下研究杂金属络合物的形态并采用光反应标记策略来确定跨膜穿梭蛋白的相互作用。(2) (如何)在没有可接触到的氧化还原事件的情况下，金属载体中的M3+是如何释放的？Fe3+被释放通过还原为Fe2+和依赖于Fe2+的蛋白质诱导的金属载体的酶降解。这个在生理条件下，感兴趣的金属化合物，GA3+，SC3+和In3+，没有可接近的氧化还原事件。我们将使用放射化学标记策略来跟踪它们由金属载体介导的摄取并识别代谢物。(3)M3+在细胞质中的去向及其对蛋白质表达的影响？非氧化还原活性金属离子到达细菌细胞质后的去向，包括它们对细菌的影响细菌蛋白的表达还不是很清楚，但却是其生长抑制活性的关键。我们会将放射化学标记策略与质谱学相结合，分离和鉴定异金属靶蛋白。我们将评估和量化接触不同异种金属后细菌代谢物的变化。金属载体复合体，这将提供有关细菌代谢变化的信息。