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.

细菌病毒与养分获得密切相关，这对于生长和增殖至关重要 病原体。金属离子构成必需的营养素，并调节细菌金属离子稳态 在主机环境中起着关键作用；然而，未结合的金属离子表现出较低的生物利用度。 例如，pH 7.4时Fe（OH）3（ksp = 6.3 x 10-38）的低溶解度将导致数量不足 铁使细菌生长，因此细菌依赖于靶向宿主的不稳定铁储备。 内源性的，亲水的金属噬菌体，使用依赖ATP的细菌跨膜内部化 穿梭。这些金属泳剂还保留了对非必需的Xenomemition离子的亲和力，并具有相同的电荷， 与必需金属离子相当的离子半径和化学硬度。例如，带有三价金属离子 与高自旋Fe3+（0.78Å）相似的离子半径，例如Ga3+（0.76Å），SC3+（0.87Å）和In3+（0.93Å） 当通过细菌铁 - 亚物种（如 肠霉素或desferioxamine。这些Xenotal不能用于所需的生物学功能。最近的 利用细菌金属稳态途径进行治疗的策略引起了人们的兴趣 在Xenotals中作为替代抗生素。 在细菌中，铁的细胞质命运以及对基因和蛋白质调节的影响是充分理解的。然而， Xenometal稳态和利用率，特别是鉴于差异pH依赖性规范行为， 仍然是基本的。为此，我们试图调查以下问题：（1）是M3+金属成绩 复合物有效地识别并在细菌机制上运输？大小，硬度和路易斯酸度 金属离子影响其配位复合物结构。与母体Fe3+复合物的显着差异 导致运输效率降低。我们将在生理学下研究Xenomemempal复杂规范 条件并采用光电反应性标记策略来识别跨膜穿梭蛋白相互作用。 （2） （如何）在没有可访问的氧化还原事件的情况下，M3+从金属泳道中释放？ Fe3+已发布 通过还原为Fe2+依赖性蛋白诱导的金属球的Fe2+和酶促降解。这 感兴趣的Xenotal，GA3+，SC3+和IN3+，在生理条件下没有可访问的氧化还原事件。 我们将采用放射化学标签策略来跟踪其金属酚介导的摄取并确定 代谢物。 （3）细胞质中M3+ Xenotal的命运是什么及其对蛋白质表达的影响？ 一旦到达细菌细胞质，非还原活性Xenotal的命运，包括它们对 细菌蛋白表达尚不清楚，但持有其生长抑制活性的关键。我们将 将放射化学标记策略与质谱分离株结合并识别Xenometal-target蛋白。 我们将在暴露于不同的Xenometal-之后评估和量化细菌代谢产物的变化 金属酚络合物将导致细菌代谢改变。