Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues
Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues
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DOI：
10.1002/cbic.201800687
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发表时间：
2019-05-15
期刊：
影响因子：
3.2
通讯作者：
Swarts, Benjamin M.
中科院分区：
文献类型：
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作者：
Fiolek, Taylor J.;Banahene, Nicholas;Swarts, Benjamin M.
分枝杆菌和杆状杆菌亚目的相关生物的特征是一种独特的外膜，称为菌膜。菌膜的生物合成是通过一个被称为菌基化的重要过程进行的，这个过程包括抗原85 （Ag85）催化的菌基酸从菌基供体海藻糖单菌酸（TMM）转移到受体碳水化合物，在某些生物体中，转移到蛋白质。我们最近描述了一种炔修饰的TMM类似物（O-AlkTMM-C7），它与click化学结合，作为完整细胞中mycolyl的化学报告，并允许对mycolyl化的菌膜成分进行代谢标记。在这里，我们描述了一个工具箱的合成和评估的tmm为基础的报告含有炔，叠氮化物，反式环烯和荧光标签。这些化合物进一步深入了解了细胞背景下mycoloyltransferases（例如Ag85s）的底物耐受性，它们通过允许一步或两步细胞标记，活细胞标记和通过四嗪连接的快速细胞标记，提供了显着扩展的实验通用性。这样的能力将促进对菌膜组成、生物合成和动力学的研究。此外，由于TMM仅由棒状杆菌代谢，因此所述探针可能对结核分枝杆菌及相关病原体的特异性检测和细胞表面工程有价值。我们还进行了实验，以确定探针掺入对mycoloyltransferase活性的依赖性，结果表明细胞标记不仅是代谢掺入（以及可能的去除）途径的功能，而且还具有跨包膜的可达性。因此，当包膜通透性可能受损时，应仔细设计和解释TMM报告的全细胞标记实验。另一方面，TMM报告者的这种特性可以作为一种方便的方法来探测包膜完整性和渗透性的变化，从而促进药物开发研究。
Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM-based reporters bearing alkyne, azide, trans-cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one- or two-step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell-surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole-cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.