Chemical Tools to Study and Exploit Bacterial Cell Wall Assembly

 描述(申请人提供)：分枝杆菌和棒状杆菌的致病物种拥有厚厚且不透水的细胞壁，通过阻止药物和探针分子进入它们的细胞质来帮助这些生物逃避检测和治疗。这种屏障的一个显著特征是由真菌酸组成的额外的疏水外膜，真菌酸共价结合到阿拉伯半乳糖(AG)多糖上，提供与细胞表面肽聚糖的联系。如果能更全面地了解生物体的整个生命周期中其组成部分是如何组装和维护的，那么查明这些外层防御系统中的弱点的努力将大大受益。该项目旨在开发专门针对和开发细胞外真菌转移酶Ag85A-C的化学工具，该酶介导真菌酸与细胞壁AG的共价结合。首先，我们将详细介绍一种策略，用荧光团共价标记细胞壁的霉菌-阿拉伯半乳糖(MAG)复合体，以实现MAG生物合成的实时可视化。我们的方法包括设计和合成内源性霉菌基供体海藻糖单菌酯(TMM)的模拟物，它携带荧光团结合的霉菌酸。这些底物将被添加到活细菌中，在那里我们将评估Ag85对它们的识别以及它们的荧光货物进入正在生长的细胞壁的情况。我们预计，这种强大的标记策略将允许对细胞壁组装的局部化和动力学进行前所未有的详细研究。最后，我们描述了一种开发Ag85触发的开启荧光探针的方法，该探针设计用于在低浓度的分枝杆菌存在的情况下进行荧光。我们预计，这种化学工具将有助于及早发现致病分枝杆菌。

 DESCRIPTION (provided by applicant): Pathogenic species of mycobacteria and corynebacteria possess a thick and impermeable cell wall that helps these organisms evade detection and treatment by denying drug and probe molecules access to their cytoplasm. A distinctive feature of this barrier is an additional hydrophobic outer membrane composed of mycolic acids, which are covalently bound to an arabinogalactan (AG) polysaccharide that provides a link to the cell surface peptidoglycan. Efforts to identify weaknesses in these outer defenses would benefit greatly from a more complete understanding of how its components are assembled and maintained throughout the life cycle of the organisms. This project aims to develop chemical tools that specifically target and exploit the extracellular mycolyltransferase enzymes Ag85A-C, which mediate the covalent attachment of mycolic acids to the cell wall AG. First, we will detail a strategy to covalently label the cell wall mycolyl-arabinogalactan (mAG) complex with fluorophores to enable the visualization of mAG biosynthesis in real time. Our approach involves the design and synthesis of mimics of the endogenous mycolyl donor trehalose monomycolate (TMM), which carry fluorophore-conjugated mycolic acids. These substrates will be added to living bacteria, where we will evaluate their recognition by Ag85 and the incorporation of their fluorescent cargo into the growing cell wall. We anticipate that this robust labeling strategy will allow the localization and dynamics of cell wall assembly to be studied in unprecedented detail. Finally, we describe an approach to develop an Ag85-triggered turn-on fluorescent probe designed to fluoresce specifically in the presence of low concentrations of mycobacteria. We envisage that such a chemical tool would facilitate the early detection of pathogenic mycobacteria.