Protein interactions regulate iron storage and utilization in bacteria

该项目的成果将填补我们对铜绿假单胞菌病原体中细菌铁代谢的基本了解的一个重大空白。这种病原体的几个菌株已经产生了多重耐药性，给医院感染的治疗带来了严峻的挑战，这些感染影响到烧伤患者、癌症患者和患有慢性肺部感染的囊性纤维化患者。目前的提议虽然是根本的，但也可能暴露出细菌铁代谢的脆弱性，可在未来开发针对细菌铁稳态的治疗方法时加以利用。该项目将在一个多学科的环境中培养研究生和本科生，该环境来自化学、生物化学、光谱学、结构生物学和微生物学等不同领域。此外，堪萨斯大学(Kansas University)的希望收获领导学院(HHL)为来自外来务农背景的高中生举办了一所暑期寄宿学院。项目主任将与人类白细胞抗原计划主任合作，组织和带领学生参观核心研究实验室，在那里学生将有机会会见核心实验室主任并与其互动。成为KU本科生的HHL学生在第一年由KU的大学助学移民计划(CAMP)组织指导。每年夏天将招募两名值得参加该夏令营项目的学生参加与该项目相关的研究活动。该项目将测试一种新的细菌铁稳态模型，即铜绿假单胞菌中两种蛋白质(BFRB和BFD)之间的相互作用使细菌细胞能够正确地感知细胞内游离铁水平的变化，并有效地将铁结合到铁利用蛋白中。BFRB是一种铁储存蛋白，BFD是从BFRB动员铁所必需的铁还蛋白。公认的细菌铁稳态模型认为，铁储存蛋白只是作为铁蓄积器发挥作用，只有当细菌细胞面临铁限制条件时，积累的铁才被动员到细胞质中。新的模型假设，BFRB：BFD的相互作用，通过促进铁通量流出BFRB，使细胞质中的游离铁和储存在BFRB中的铁之间实现动态平衡，这对细胞感知游离铁水平的变化并对其做出反应至关重要。修订后的模型还表明，BFRB的铁动员是有效地将铁结合到铁利用蛋白中所必需的，因此抑制BFRB：BFD的相互作用将导致铁结合到铁利用酶中的效率低下，这将对依赖于含铁蛋白和酶的重要生理过程产生不利影响。这条建议的途径不同于公认的模型中的途径，在该模型中，利用铁的蛋白质的铁的来源被假设为胞质中的游离铁。研究人员还发现了一个抑制BFRB：BFD相互作用的小分子探针，并获得了该分子增强一些现有抗生素的杀伤力的证据。因此，该项目还旨在从根本上了解BFRB：BFD相互作用的抑制剂如何增强抗生素的活性。这些研究的指导思想是，由BFRB：BFD相互作用的小分子抑制剂引起的BFRB中不可逆转的铁积累，严重限制了支持抵御抗生素诱导的应激所需的关键代谢过程所需的细胞内铁。

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.