Chemical Tools to Study and Exploit Bacterial Cell Wall Assembly

 描述（由申请方提供）：分枝杆菌和棒状杆菌的致病菌种具有厚且不可渗透的细胞壁，通过阻止药物和探针分子进入其细胞质，有助于这些生物体逃避检测和治疗。这种屏障的一个显著特征是由分枝菌酸组成的额外疏水外膜，分枝菌酸与阿拉伯半乳聚糖（AG）多糖共价结合，提供与细胞表面肽聚糖的连接。更全面地了解这些外部防御系统的组成部分是如何在生物体的整个生命周期中组装和维持的，将大大有助于确定这些外部防御系统的弱点。该项目旨在开发专门针对和利用细胞外分枝菌酰转移酶Ag 85 A-C的化学工具，该酶介导分枝菌酸与细胞壁AG的共价连接。首先，我们将详细介绍一种策略，共价标记细胞壁分枝菌酰-阿拉伯半乳聚糖（mAG）复合物与荧光团，使mAG生物合成的可视化在真实的时间。我们的方法涉及设计和合成的模拟物的内源性分枝菌酰供体海藻糖monomycolate（TMM），携带荧光团共轭分枝菌酸。这些底物将被添加到活细菌中，在那里我们将评估它们被Ag 85识别并将其荧光货物掺入生长的细胞壁中。我们预计，这种强大的标记策略将允许细胞壁组装的本地化和动态进行研究，在前所未有的细节。最后，我们描述了一种方法来开发一种Ag 85触发的开启荧光探针，其设计用于在低浓度分枝杆菌存在下特异性地发出荧光。我们设想这样的化学工具将有助于早期发现致病性分枝杆菌。

 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.