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.

细菌的毒力与营养获取密切相关，营养获取对于生长和增殖至关重要 病原体。金属离子构成细菌必需的营养物质，并调节金属离子的体内平衡 在宿主环境中起关键作用;然而，未结合的必需金属离子表现出低生物利用度。 例如，Fe（OH）3在pH 7.4下的低溶解度（Ksp = 6.3 X 10-38）将导致Fe（OH）3的量不足。 铁是细菌生长所必需的，因此细菌依赖于靶向宿主不稳定的铁储备，通过合成 内源性亲水性金属载体，通过ATP依赖性细菌跨膜作用内化 穿梭机这些金属载体还保持对具有相同电荷的非必需的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，三价金属离子与 与高自旋Fe 3+（0.78 μ m）类似离子半径，如Ga 3+（0.76 μ m）、Sc 3+（0.87 μ m）和In 3+（0.93 μ m）， 当与细菌铁-金属载体如 肠杆菌素或去铁胺。这些异金属不能用于所需的生物功能;最近 利用细菌金属稳态途径来递送治疗剂的策略引起了人们的新兴趣 作为替代抗生素。 在细菌中，铁的细胞质命运以及对基因和蛋白质调控的影响是众所周知的;然而， 异金属的稳态和利用，特别是根据不同的pH值依赖的物种形成行为， 仍然是基本的。为此，我们试图研究以下问题：（1）M3+是金属载体吗 复合物有效地识别和运输通过细菌膜？粒度、硬度和刘易斯酸度 金属离子对配合物结构的影响。与母体Fe 3+络合物的显著差异 导致输送效率降低。我们将在生理条件下研究异金属络合物的形态 条件，并采用光反应性标记策略来鉴定跨膜穿梭蛋白相互作用。（二） (How)M3+从金属载体的释放是否在没有可接近的氧化还原事件的情况下进行？Fe 3+释放 通过还原为Fe 2+和由Fe 2+依赖性蛋白诱导的金属载体的酶促降解。的 感兴趣的异金属Ga 3+、Sc 3+和In 3+在生理条件下不具有可接近的氧化还原事件。 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，并鉴定 代谢物。(3)M3+异金属在细胞质中的命运及其对蛋白质表达的影响是什么？ 非氧化还原活性的异金属的命运，一旦它们到达细菌细胞质，包括它们对细菌细胞质的影响。 细菌蛋白质的表达还没有被很好地理解，但却掌握着它们的生长抑制活性的关键。我们将 联合收割机放射化学标记策略与质谱分离和鉴定氙靶蛋白。 我们将评估和定量暴露于不同的异金属后细菌代谢物的变化， 金属复合物，这将告知改变细菌代谢。