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

• 批准号: 10847170
• 负责人: Eszter Boros
• 金额: $ 38.87万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10847170
• 关键词: Tracking; elucidation; modulation; xenometal; homeostasis

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.

细菌毒力与营养获取密切相关，这对于生长和增殖至关重要 金属离子构成病原体必需的营养物质，以及细菌金属离子稳态的调节。 在宿主环境中起着关键作用；然而，未结合的必需金属离子表现出较低的生物利用度。 例如，Fe(OH)3 (Ksp = 6.3 x 10-38) 在 pH 7.4 时的溶解度较低，会导致 Fe(OH)3 的溶解度不足。 铁供细菌生长，因此依靠细菌通过合成来靶向宿主的不稳定铁储备 使用 ATP 依赖性细菌跨膜内化的内源性亲水性金属载体 这些金属载体还保留对具有相同电荷的非必需异金属离子的亲和力， 与基本金属离子相当的离子半径和化学硬度，例如，具有的三价金属离子。 与高自旋 Fe3+ (0.78 Å) 类似的离子半径，例如 Ga3+ (0.76 Å)、Sc3+ (0.87 Å) 和 In3+ (0.93 Å) 当与细菌铁金属载体协调时，转运至细菌周围和细胞质，例如 这些异金属不能用于近期所需的生物学功能； 利用细菌金属稳态途径提供治疗的策略引起了人们的新兴趣 在异金属中作为替代抗生素。 然而，在细菌中，铁的细胞质命运以及对基因和蛋白质调节的影响是众所周知的。 异种金属稳态和利用，特别是考虑到不同的 pH 依赖性形态行为， 为此，我们试图研究以下问题：（1）M3+-金属团是吗？ 复合物能有效识别并穿过细菌膜吗？尺寸、硬度和路易斯酸度？ 金属离子的影响其配位络合物结构与母体 Fe3+ 络合物的显着差异。 我们将研究生理条件下异种金属复合物的形态。 条件并采用光反应标记策略来识别跨膜穿梭蛋白相互作用 (2)。 （如何）在没有可及的氧化还原事件释放 Fe3+ 的情况下，M3+ 是否会从金属载体中释放？ 通过还原为 Fe2+ 以及由 Fe2+ 依赖性蛋白诱导的金属团的酶促降解。 感兴趣的异种金属 Ga3+、Sc3+ 和 In3+ 在生理条件下不发生氧化还原事件。 我们将采用放射化学标记策略来跟踪它们的金属载体介导的摄取并识别 (3) M3+ 异种金属在细胞质中的命运及其对蛋白质表达的影响是什么？ 非氧化还原活性异种金属一旦到达细菌细胞质后的命运，包括它们对 细菌蛋白的表达尚不清楚，但我们将掌握其生长抑制活性的关键。 将放射化学标记策略与质谱分离和鉴定异种金属靶蛋白相结合。 我们将评估和定量暴露于不同异金属后细菌代谢物的变化 金属载体复合物，它将告知细菌代谢的改变。