Dissecting the structure and function of the Pdu microcompartment in Salmonella

描述（由申请方提供）：细菌微区室是由代谢酶组成的大型亚细胞结构，这些代谢酶封装在由多个亚基构建的蛋白质壳内。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，并且似乎包含独特的机制和结构原则。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，很少有人知道这是如何发生在机械水平。该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区室的三维结构。沙门氏菌系统是无与伦比的知识和工具，可用于微区室的机制研究。拟议的研究结合联合收割机遗传，生物物理和结构的方法来阐明沙门氏菌Pdu微区室的细胞功能的机制水平。提出了三个具体目标：1。确定微区室壳蛋白和酶的结构; 2.阐明Pdu微区室中的生物化学相互作用和高阶结构;以及3.进行微区室功能突变分析。结构将通过X射线晶体学，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、标记研究和晶体学。功能和机制的见解将来自结构指导的诱变结合生长研究，酶和运输试验，反向双杂交分析。完成拟议的调查将阐明沙门氏菌pdu微区室的机制和结构原则。这将提供对细菌微区室的一般了解。此外，由于细菌微区室在许多微生物（包括几种人类病原体）中起着关键的代谢作用，因此拟议的研究可能最终导致干扰致病过程的新机会。公共卫生相关性：细菌微区室在许多微生物中起着关键的代谢作用，包括几种人类病原体，但目前对它们的了解非常少。该提案旨在了解Pdu微区室在人类病原体沙门氏菌鼠伤寒血清型中的功能。这些研究将导致对这些重要的细菌细胞器的更深入的基础和机制理解，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.