Protein interactions regulate iron storage and utilization in bacteria

该项目的成果将填补我们对病原菌铜绿假单胞菌细菌铁代谢的基本认识的重大空白。这种病原体的一些菌株已经产生了多药耐药性，给烧伤患者、癌症患者和慢性肺部感染的囊性纤维化患者的医院感染治疗带来了严峻挑战。目前的提议，虽然本质上是基本的，但也可能暴露细菌铁代谢的脆弱性，可以在未来开发针对细菌铁稳态的疗法中加以利用。该项目将在化学、生物化学、光谱学、结构生物学和微生物学等不同领域的多学科环境中培养研究生和本科生。此外，堪萨斯大学（KU）的丰收希望领导学院（HHLA）为来自移民农场工作背景的高中生举办了一个暑期住宿学院。项目主任将与HHLA项目主任合作，组织并带领学生参观核心研究实验室，学生将有机会与核心实验室主任见面并互动。成为堪萨斯大学本科生的HHLA学生在第一年由堪萨斯大学的大学援助移民计划（CAMP）组织指导。每年夏天将从CAMP项目中招募两名优秀的学生参加与该项目相关的研究活动。该项目将测试细菌铁稳态的新模型，即铜绿假单胞菌（P. aeruginosa）中两种蛋白质（BfrB和Bfd）之间的相互作用使细菌细胞能够正确感知细胞内游离铁水平的变化，并有效地将铁纳入利用铁的蛋白质中。BfrB是一种铁储存蛋白，而Bfd是BfrB动员铁所需的铁氧还蛋白。公认的细菌铁稳态模型假设，铁储存蛋白的功能只是作为铁积累器，积累的铁只有在细菌细胞面临铁限制条件时才被动员到细胞质中。新模型假设BfrB:Bfd相互作用，通过促进BfrB的铁通量，实现细胞质中游离铁和BfrB中储存的铁之间的动态平衡，这对于细胞感知和反应游离铁水平的变化至关重要。修正后的模型还表明，铁需要从BfrB中动员，才能有效地将铁并入利用铁的蛋白质中，因此抑制BfrB:Bfd相互作用将导致铁低效地并入利用铁的酶中，这将对依赖含铁蛋白质和酶的重要生理过程产生不利影响。这个提议的路径不同于在公认的模型中，其中利用铁的蛋白质的铁来源被认为是胞质溶胶中的游离铁。研究人员还发现了一种抑制BfrB:Bfd相互作用的小分子探针，并获得证据表明该分子增强了一些现有抗生素的杀伤活性。因此，该项目还旨在获得对BfrB:Bfd相互作用抑制剂如何增强抗生素活性的基本理解。这些研究的指导思想是，铁在BfrB中的不可逆积累是由BfrB:Bfd相互作用的小分子抑制剂引起的，严重限制了细胞内铁的需要，而细胞内铁需要支持关键的代谢过程，以抵御抗生素诱导的应激。

The project outcomes will fill a major gap in our fundamental understanding of bacterial iron metabolism in the pathogen Pseudomonas aeruginosa. Several strains of this pathogen have developed multidrug resistance, causing severe challenges for the treatment of hospital infections that affect burn victims, cancer patients and cystic fibrosis patients with chronic lung infections. The present proposal, while fundamental in nature, is also likely to expose bacterial iron metabolism as a vulnerability that can be exploited in future development of therapies that target bacterial iron homeostasis. The project will train graduate and undergraduate students in a multidisciplinary environment that draws from the diverse fields of chemistry, biochemistry, spectroscopy, structural biology and microbiology. In addition, the Harvest of Hope Leadership Academy (HHLA) at the University of Kansas (KU) hosts a summer residential academy for high school students from migrant farm-work background. The Project Director, in collaboration with the HHLA Program Director, will organize and lead students on tours of Core Research laboratories, where students will have the opportunity to meet and interact with the Core Laboratory Directors. HHLA students who become KU undergraduates are mentored during their first year by the College Assistance Migrant Program (CAMP) organization at KU. Two deserving students from the CAMP program will be recruited each summer to participate in research activities related to the project.The project will test a new model for bacterial iron homeostasis, whereby the interactions between two proteins in P. aeruginosa (BfrB and Bfd) enable the bacterial cells to correctly sense changes in intracellular free iron levels and to efficiently incorporate iron into iron-utilizing proteins. BfrB is an iron storage protein and Bfd is a ferredoxin required for iron mobilization from BfrB. The accepted model of bacterial iron homeostasis assumes that iron storage proteins function simply as iron accumulators and that accumulated iron is mobilized to the cytosol only when bacterial cells face iron-limiting conditions. The new model posits that the BfrB:Bfd interaction, by facilitating iron flux out of BfrB, enables a dynamic equilibrium between free iron in the cytosol and iron stored in BfrB, which is crucial for the cells to sense and react to changes in free iron levels. The revised model also suggests that iron mobilization from BfrB is required for the efficient incorporation of iron into iron-utilizing proteins, so that inhibition of the BfrB:Bfd interaction will result in inefficient iron incorporation into iron-utilizing enzymes, which will adversely affect important physiological process that depend on iron-containing proteins and enzymes. This proposed path is different from that in the accepted model, where the source of iron for iron-utilizing proteins is assumed to be the free iron in the cytosol. The investigators have also discovered a small molecule probe that inhibits the BfrB:Bfd interaction, and obtained evidence that this molecule enhances the killing activity of some existing antibiotics. Hence, the project also aims to obtain a fundamental understanding how the antibiotic activity is enhanced by inhibitors of the BfrB:Bfd interaction. These studies will be guided by the idea that irreversible accumulation of iron in BfrB, which is caused by the small molecule inhibitor of the BfrB:Bfd interaction, severely restricts the intracellular iron needed to support crucial metabolic processes required to fend antibiotic-induced stress.