Phosphorylated Carbohydrate Transport Across the Bacterial Cell Envelope

细菌依赖于从宿主获得一系列不同的营养物质来进行成功的生长、定植或致病。共生和/或致病细菌与宿主细胞竞争从金属到维生素的各种底物。由于这种竞争可以影响感染的结果，病原体已经发展了各种营养吸收机制来提高它们的竞争力，从而提高它们定植和高效感染宿主的能力。我的研究计划的长期目标是通过分离膜蛋白并使用X射线结晶学和其他生物物理工具的独特组合来进行原子分辨率结构和功能表征，从而阐明细菌营养吸收的分子基础，这些工具包括微尺度热电泳法和生物层干涉法、表面等离子体共振和等温滴定热量计。 我的NSERC研究重点是通过结合蛋白依赖转运体(BPDT)的营养吸收机制，BPDT针对不同的配体，如金属离子、氨基酸和糖，被认为是许多细菌感染的毒力决定因素。我在这项提案中概述的短期目标是破译AfuABC的机制，AfuABC是一种结合蛋白依赖的转运体，我们确定它负责主动摄取磷酸化碳水化合物。我们的结构洞察力将提供从宿主中清除营养的分子基础，以及这些细菌如何在复杂的环境中竞争营养。 目的1.鉴定底物特异性和转运的AfuABC同系物和类似物。 目的2.通过晶体结构、生长分析和生物分子相互作用分析AfuABC转运磷酸糖的机制，探讨AfuA和AfuBC之间的相互作用。 目的3.确定转运蛋白在致病机制中的作用以及这些细菌获得底物的来源。 这些目标将导致对细菌病原体获取磷糖养分的关键理解，以及这一过程如何在其环境中提供竞争优势。此外，这项研究计划将提供研究膜蛋白及其相互作用的工具，为我的3名本科生、6名研究生和2名博士后研究员提供理想而独特的培训环境，其中3人将参与AIMS 1和2中概述的微生物学、生物化学和生物物理方法的研究。为了与我迄今成功培养的高素质人才保持一致，我的受训人员将有机会为高影响力的出版物做出贡献，并将获得膜蛋白结构生物学、微生物学和生物化学方面的广泛专业知识，为他们在基础科学研究和生物技术领域的职业生涯做好准备。

Bacteria depend on the acquisition of a diverse set of nutrients from their hosts to engage in successful growth, colonization or pathogenesis. Commensal and/or pathogenic bacteria compete with host cells for substrates ranging from metals to vitamins. As this competition can influence the outcome of infection, pathogens have developed a variety of nutrient uptake mechanisms to increase their competitiveness and hence their ability to colonize and productively infect their hosts. The long-term objective of my research programs is to elucidate the molecular basis of bacterial nutrient uptake by isolating membrane proteins and performing atomic resolution structural and functional characterization using a unique combination of X-ray crystallography and other biophysical tools such as microscale thermophoresis and biolayer interferometry, surface plasmon resonance and isothermal titration calorimetry. My NSERC research focuses on nutrient uptake mechanisms via the binding-protein dependent transporters (BPDTs), which target diverse ligands such as metal ions, amino acids and sugars and are recognized as virulence determinants in many bacterial infections. My short-term objectives outlined in this proposal are to decipher the mechanism of AfuABC a binding protein dependent transporter that we determined is responsible for active uptake of phosphorylated carbohydrates. Our structural insights will provide the molecular basis of nutrient scavenging from the host and how these bacteria compete for nutrients in complex environments. Aim 1. Characterize the identify AfuABC homologs and paralogs for substrate specificity and transport. Aim 2. Dissect the mechanism of transport of phospho-sugars by AfuABC using crystal structures, growth assays and biomolecular interactions to probe the interaction between the AfuA and AfuBC components of the uptake system. Aim 3. Determine the role of transporters in the pathogenesis and the source of the substrates that are acquired by these bacteria. These aims will lead to a critical understanding of phospho-sugar nutrient acquisition by bacterial pathogens and how this process provides a competitive advantage in their environment. In addition, this research program will provide tools to investigate membrane proteins and their interactions, providing an ideal and unique training environment for my 3 undergraduates, 6 graduate students and 2 postdoctoral fellows, 3 of whom will be involved in conducting the microbiology, biochemistry and biophysical methods outline in aims 1 and 2. In keeping with my successful training of highly qualified personnel to date, my trainees will have the opportunity to contribute to high-impact publications and will gain broad expertise in membrane protein structural biology, microbiology, and biochemistry, preparing them for careers in basic scientific research and biotechnology.