Photoactivatable glycolipid probes for identifying mycolate–protein interactions in live mycobacteria,Journal of the American Chemical Society

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Photoactivatable glycolipid probes for identifying mycolate–protein interactions in live mycobacteria
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2020-04-15 , DOI: 10.1021/jacs.0c01065
Herbert W Kavunja ₁ , Kyle J Biegas ₁ , Nicholas Banahene ₁ , Jessica A Stewart ₁ , Brent F Piligian ₁ , Jessica M Groenevelt ₁ , Caralyn E Sein ₂ , Yasu S Morita _{2,

3} , Michael Niederweis ₄ , M Sloan Siegrist _{2,

3} , Benjamin M Swarts ₁

Affiliation

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-crosslinking and click chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it: (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

中文翻译：

用于鉴定活分枝杆菌中分枝菌酸盐-蛋白质相互作用的光活化糖脂探针

分枝杆菌具有独特的富含糖脂的外膜，即菌膜，它是结核病药物开发的关键目标。然而，与菌膜相关的蛋白质或参与其代谢和宿主相互作用的蛋白质尚未得到充分表征。为了促进菌膜相关蛋白的研究，我们开发了可光活化的海藻糖单分枝菌酸酯类似物，其通过代谢方式融入活分枝杆菌的菌膜中，从而能够对分枝菌相互作用蛋白进行体内光交联和点击化学介导的分析。当在耻垢分枝杆菌中采用定量蛋白质组学时，该策略富集了超过 100 种蛋白质，包括菌膜孔蛋白 (MspA)、几种具有已知菌膜合成或重塑功能的蛋白质（CmrA、MmpL3、Ag85、Tdmh）以及许多候选霉菌酸盐相互作用蛋白。我们的方法具有高度通用性，因为它：（i）利用点击化学来实现灵活的蛋白质功能化； (ii) 原则上可应用于任何分枝杆菌物种，以鉴定与分枝菌酸盐相互作用的内源细菌蛋白或宿主蛋白；（iii）可以潜在地扩展到研究蛋白质与其他分枝杆菌脂质的相互作用。该工具有望帮助阐明与菌膜相关的基本生理和病理过程，并可能揭示新的诊断和治疗靶点。

更新日期：2020-04-15

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