Understanding trehalose synthesis and utilization in mycobacteria

描述(由申请人提供)：本提案的首要目标是更好地了解分枝杆菌中海藻糖生物合成、利用和再循环的关键步骤。二糖海藻糖对分枝杆菌生理学的多个方面都很重要，并已被证明是生存所必需的，导致海藻糖单菌酸酯生产和出口的下游利用途径也是如此。回收用于构建外膜的海藻糖对结核分枝杆菌的毒力非常重要，并用于延长胞质葡聚糖的长度。这个研究项目有三个不同的方面，都与海藻糖代谢有关。首先，我们将确定促进底物结合和催化的结核分枝杆菌GlgE的结构特征，作为了解其在合成分枝杆菌葡聚糖中的功能的基础。由于GlgE的抑制促进了对结核分枝杆菌的快速杀伤，因此将合成基于机制的抑制剂，并结合稳态动力学使用，以更好地了解酶的作用机制。将进行X射线结晶学研究，以表征与GlgE底物的相互作用，这将形成针对GlgE的药物开发的基础。这项研究的第二部分旨在进一步表征抗结核药物依贝塞伦杀死分枝杆菌的机制。我们已经证明，ebselen通过破坏酶活性部位并使其失活的共价修饰来强烈抑制抗原85C。在体内和体外进行的实验将确定细菌培养中哪些分枝杆菌蛋白被ebselen修饰。第三个目标将描述催化从头合成海藻糖生物合成途径的最后一步--海藻糖磷酸磷酸酶2的结构-功能关系。稳态动力学将用于研究活性部位突变和体外抑制的影响。非活性突变体也将用于平衡结合研究，以更好地了解底物的选择性。这些信息将为在体内进行的研究提供信息，并确定TPP2是否为有效的药物靶点。这些研究的结果将被用来促进我们对使用海藻糖的代谢途径的了解。用于形成菌膜的积木的生物合成，真菌酸和海藻糖单菌酯，是一线和二线抗结核药物的已知靶点。因此，预计进一步定义生物合成 通向海藻糖单菌酸酯的途径和将真菌酸附在菌膜上的酶的特征将为抗结核药物的开发提供新的见解。预计这项研究将扩大现有的结核分枝杆菌药物靶点，将海藻糖生物合成和利用途径中的酶包括在内。

DESCRIPTION (provided by applicant): The overarching goal of this proposal is to better understand key steps in the biosynthesis, utilization and recycling of trehalose in mycobacteria. The disaccharide trehalose is important for multiple aspects of mycobacterial physiology and has been shown to be essential for viability as is the downstream utilization pathway that leads to trehalose monomycolate production and export. Recycling of the trehalose used to build the outer membrane is important for M. tuberculosis virulence and is used to elongate a cytosolic glucan. This research project possesses 3 separate aspects that are all related to trehalose metabolism. First, we will identify structural features of M. tuberculosis GlgE that promote substrate binding and catalysis as a basis for understanding its function in synthesizing the mycobacteria glucan. Since inhibition of GlgE promotes rapid killing of M. tuberculosis, mechanism based inhibitors will be synthesized and used in conjunction with steady-state kinetics to better understand the enzyme mechanism. X-ray crystallographic studies will be performed to characterize interactions with GlgE substrates, which will form the basis for drug-development targeting GlgE. The second portion of this study aims to further characterize the mechanism of mycobacterial killing by the anti-tubercular drug ebselen. We have shown that ebselen strongly inhibits Antigen 85C through a covalent modification that disrupts the enzyme active site and inactivates it. Experiments performed in vivo and ex vivo will identify which mycobacterial proteins are modified by ebselen in a bacterial culture. The third aim will characterize the structure-function relationship of the enzyme catalyzing the final step in the de novo trehalose biosynthetic pathway, trehalose phosphate phosphatase 2. Steady-state kinetics will be used to study the effects of active site mutations and inhibition in vitro. Inactive mutant will also be used in equilibrium binding studies to better understand substrate selectivity. This information will inform studies performed in vivo and determine if TPP2 is a valid drug target. The results from these studies will be used to advance our knowledge of the metabolic pathways that use trehalose. The biosynthesis of the building blocks used to form the mycomembrane, mycolic acids and trehalose monomycolate, are known targets of first and second-line anti-tubercular drugs. Therefore, it is expected that further defining the biosynthetic pathway leading to trehalose monomycolate and characterizing the enzymes that attach mycolic acids to the mycomembrane will offer new insights for anti-tubercular drug development. It is expected that this study will extend the available Mycobacterium tuberculosis drug targets to include enzymes in the trehalose biosynthetic and utilization pathways.