Dissecting the structure and function of the Pdu microcompartment in Salmonella

描述(申请人提供)：细菌微室是由代谢酶组成的大的亚细胞结构，包裹在由多个亚基组成的蛋白质外壳中。它们在细菌中广泛存在，功能多样，发挥重要的代谢作用，与发病机制有关，并似乎包含独特的机制和结构原理。它们的功能是隔离和调节某些代谢途径中发现的有毒或挥发性中间体的产生。然而，对于这是如何在机制层面上发生的，我们知之甚少。这项拟议研究的长期目标是阐明沙门氏菌降解1，2-丙二醇的分子原理并建立微室的三维结构。沙门氏菌系统在可用于微格机械研究的知识和工具方面是无与伦比的。拟议的研究结合了遗传学、生物物理学和结构学方法，在机制水平上阐明了沙门氏菌PDU微隔室的细胞功能。提出了三个具体的目标：1.确定微室壳蛋白和酶的结构；2.阐明PDU微室中的生化相互作用和高阶结构；3.对微室功能进行突变分析。结构将通过X射线结晶学、生物物理学和计算方法进行研究和分析。相互作用研究将包括双杂交分析、标记研究和结晶学。功能和机制的洞察将来自结构引导的突变，结合生长研究、酶和运输分析以及反向双杂交分析。完成拟议的调查将阐明沙门氏菌PDU微格的机理和结构原理。这将提供对细菌微隔室的一般见解。此外，由于细菌微室在许多微生物中发挥着关键的代谢作用，包括几种人类病原体，拟议的研究可能最终会带来干扰致病过程的新机会。与公共卫生相关：细菌微室在许多微生物中发挥着关键的代谢作用，包括几种人类病原体，但目前对它们的了解非常少。这项建议试图了解PDU微区如何在人类病原体--鼠伤寒沙门氏菌中发挥作用。这些研究将导致对这些重要细菌细胞器的更深层次的基础和机制的了解，并最终为干扰致病过程提供新的机会。

DESCRIPTION (provided by applicant): Bacterial microcompartments are large subcellular structures composed of metabolic enzymes encapsulated within a protein shell built from multiple subunits. They are widespread among bacteria, functionally diverse, play vital metabolic roles, are linked to pathogenesis, and appear to incorporate unique mechanistic and structural principles. Their function is to sequester and regulate the production of toxic or volatile intermediates found in certain metabolic pathways. However, little is known about how this is occurs at the mechanistic level. The long-term goal of the proposed research is to elucidate the molecular principles and to build up a 3-dimensional structure of the microcompartments involved in 1,2-propanediol degradation by Salmonella. The Salmonella system is unmatched with regard to the knowledge and tools available for mechanistic studies of microcompartments. The proposed studies combine genetic, biophysical, and structural methods to elucidate the cellular function of the Salmonella Pdu microcompartment at a mechanistic level. Three specific aims are proposed: 1. determine the structures of the microcompartment shell proteins and enzymes; 2. elucidate biochemical interactions and higher-order architecture in the Pdu microcompartment; and 3. conduct mutational analysis of microcompartment function. Structures will be investigated and analyzed by x-ray crystallography, biophysical, and computational methods. Interactions studies will include two-hybrid analyses, labeling studies, and crystallography. Functional and mechanistic insights will be derived from structure-guided mutagenesis in conjunction with growth studies, enzyme and transport assays, and reverse two-hybrid analyses. Completion of the proposed investigations will elucidate the mechanistic and structural principles of the Salmonella pdu microcompartment. This will provide general insights into bacterial microcompartments. Furthermore, since bacterial microcompartments play critical metabolic roles in many microbes, including several human pathogens, the proposed studies may ultimately lead to new opportunities for interfering with pathogenic processes. PUBLIC HEALTH RELEVANCE: Bacterial microcompartments play critical metabolic roles in many microbes, including several human pathogens, but they are very poorly understood at the present time. This proposal seeks to understand how the Pdu microcompartment functions in the human pathogen, Salmonella enterica serovar Typhimurium. The studies will lead to a deeper fundamental and mechanistic understanding of these important bacterial organelles, and ultimately to new opportunities for interfering with pathogenic processes.