Protein-Protein Interactions Involved in Siderophore Biosynthesis and Secretion

参与铁载体生物合成和分泌的蛋白质-蛋白质相互作用

• 批准号: RGPIN-2017-04796
• 负责人: Pawelek, Peter
• 金额: $ 1.89万
• 依托单位: Concordia University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=661774
• 关键词: Protein; Interactions; Involved; Siderophore; Biosynthesis

Most metabolic processes that support life require protein-protein interactions. Furthermore, metabolic enzymes that participate in such processes are often clustered into large complexes known as metabolons. Organization of enzymes into metabolons can enhance the efficiency of metabolic processes and can facilitate membrane localization. My laboratory studies the role of protein-protein interactions in siderophore-mediated bacterial iron uptake. Siderophores are small-molecule iron chelators that bacteria synthesize and secrete to obtain scarce iron from extracellular environment. Little is currently known about how the enzymes involved in siderophore biosynthesis are organized within a bacterial cell. We now have evidence that the enzymes involved in the biosynthesis of the E. coli siderophore enterobactin are organized as a metabolon in E. coli cells. We propose that interactions between proteins involved in enterobactin biosynthesis and secretion (i) enhance metabolic flux via substrate channeling and (ii) anchor the enterobactin metabolon to the E. coli inner membrane to facilitate secretion via direct interaction between the metabolon and an efflux transporter. We will build on our successful efforts to fully characterize the nature of interactions between enterobactin biosynthetic (Ent) enzymes using biophysical and structural approaches. We will also investigate how such protein interactions may enhance metabolic flux via substrate channeling. Knowledge gained from these experiments will allow us to identify molecular targets to disrupt Ent protein interactions and substrate channeling processes. We will study the effects of such disruptions on bacterial growth and siderophore secretion. Some E. coli strains glycosylate enterobactin prior to its secretion in order to evade host immune systems. We will study IroB, the enterobactin glycosyltransferase, to understand how its functions may be coordinated with the Ent biosynthetic machinery. In addition to our biophysical and structural approaches, we will investigate how the Ent biosynthetic machinery is localized within E. coli cells. An in vivo crosslinking approach will be used to identify protein partners in contact with the efflux transporter EntS, and to identify higher-order Ent protein complexes. In collaboration with microscopy experts, we will also use super-resolution microscopy and FRET microscopy to determine co-localization of Ent proteins in intact E. coli cells. The research proposed in this application will identify targets for either disruption or enhancement of siderophore-mediated iron uptake in E. coli cells. Small-molecule disruptors of Ent-Ent and Ent-IroB protein interactions may lead to novel antibiotics to protect poultry and livestock. Enhancement of these processes could lead to the engineering of novel bacterial strains for sustainable biomining of iron and other metals.

大多数支持生命的代谢过程需要蛋白质-蛋白质相互作用。此外，参与这些过程的代谢酶通常聚集成称为代谢子的大复合物。将酶组织成代谢子可以提高代谢过程的效率，并且可以促进膜定位。我的实验室研究蛋白质-蛋白质相互作用在铁载体介导的细菌铁吸收中的作用。铁载体是细菌合成和分泌的小分子铁螯合剂，用于从细胞外环境中获取稀缺的铁。目前对铁载体生物合成中涉及的酶在细菌细胞内是如何组织的知之甚少。我们现在有证据表明参与E.大肠杆菌铁载体肠杆菌素在E. coli细胞。我们认为参与肠杆菌素生物合成和分泌的蛋白质之间的相互作用（i）通过底物通道增强代谢通量和（ii）将肠杆菌素代谢子锚在E.大肠杆菌内膜，以促进分泌通过直接相互作用之间的代谢和外排转运。我们将建立在我们成功的努力，充分利用生物物理和结构方法肠杆菌素生物合成（Ent）酶之间的相互作用的性质。我们还将研究这些蛋白质的相互作用如何通过底物通道增强代谢通量。从这些实验中获得的知识将使我们能够确定分子靶点，以破坏Ent蛋白相互作用和底物通道过程。我们将研究这种干扰对细菌生长和铁载体分泌的影响。一些E。大肠杆菌菌株在肠杆菌素分泌之前将其糖基化，以逃避宿主免疫系统。我们将研究肠杆菌素糖基转移酶IroB，以了解其功能如何与Ent生物合成机制协调。除了我们的生物物理和结构的方法，我们将研究如何在E。大肠杆菌细胞。体内交联方法将用于鉴定与外排转运蛋白EntS接触的蛋白质伴侣，并鉴定高阶Ent蛋白复合物。与显微镜专家合作，我们还将使用超分辨率显微镜和FRET显微镜来确定Ent蛋白在完整E.大肠杆菌细胞。本申请中提出的研究将确定在E.大肠杆菌细胞。Ent-Ent和Ent-IroB蛋白相互作用的小分子干扰物可能会导致新的抗生素来保护家禽和牲畜。这些过程的增强可能会导致新的细菌菌株的工程可持续的铁和其他金属的生物采矿。