大肠杆菌细胞壁肽聚糖的化学修饰及荧光标记
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国家自然科学基金(31770862,21778074)


Chemical modification and fluorescence labeling of Escherichia coli cell wall peptidoglycan
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    摘要:

    [目的] 发展一种活细菌细胞壁荧光标记方法,为后期研究细菌肽聚糖的生物合成和代谢规律以及其与细菌感染致病的关系提供新的工具。[方法] 对细菌肽聚糖的生物合成前体N-乙酰葡萄糖胺-1-磷酸(GlcNAc-1-P)进行化学修饰,设计并合成含有叠氮基的GlcNAc-1-P类似物(化合物5:Ac3GlcNAz-1-P)。将该类似物与细菌共同孵育,使其作为探针进入细菌肽聚糖天然合成途径。之后提取并酶解肽聚糖组分,用红外光谱(FTIR)和液质联用(LC/MS)检测探针是否通过代谢进入细菌肽聚糖结构中。同时用外源荧光素对代谢掺入细菌肽聚糖中的探针进行染色。在激光共聚焦显微镜下观察对活细菌的荧光标记效果。[结果] 通过四步有机合成反应,以79%的总收率成功获得了化合物5。将大肠杆菌(Escherichia coli BL21)作为模式菌株与化合物5共孵育后,其肽聚糖组分的LC/MS和FTIR分析结果均显示探针可以被细菌利用并被代谢掺入到肽聚糖结构中。激光共聚焦显微镜观察结果显示,荧光素可以高效标记表面携带有生物正交探针的大肠杆菌。[结论] 设计合成了一种新型探针,可用于活细菌成像,为深入研究细菌肽聚糖的生物学功能及其与细菌感染致病的关系提供了一种简便的方法。

    Abstract:

    [Objective] The objective of this research is to develop a new approach for labelling the cell wall peptidoglycan (PG) of living bacteria and provide a simple and convenient tool for the study of PG biosynthesis and metabolism as well as its role in the infection and pathogenesis. [Methods] N-Acetylglucosamine 1-phosphate (GlcNAc-1-P) is a general and essential precursor of PG biosynthesis in eubacteria. We designed and synthesized an analogue of GlcNAc-1-P (designated as Ac3GlcNAz-1-P) that carries an azido group for the fluorescence labelling of PG in bacteria. Escherichia coli BL21 were used as the model bacteria and were cultured in the presence of Ac3GlcNAz-1-P to enable the metabolic incorporation of this compound into the PG biosynthetic pathway. Subsequently the PG component was isolated from these pretreated bacteria and characterized by transform infrared spectroscopy (FTIR) and liquid chromatography-high resolution mass spectroscopy (LC-MS). The labelling of bacterial PG was performed by the incubation of the pretreated bacteria with an alkyne-functionalized fluorescent dye (DBCO-Cy3), which could directly couple to azido groups through click reaction. Fluorescence was probed by confocal fluorescence microscopy (CFM). [Results] Ac3GlcNAz-1-P was successfully synthesized in four steps from D-glucosamine hydrochloride with a total yield of 79%. LC-MS and FTIR analysis of the PG extracts from E. coli pretreated with Ac3GlcNAz-1-P revealed the presence of azide-labelled sugar units in the PG structure. CFM observation confirmed that the pretreated bacteria could be stained by DBCO-Cy3 selectively and effectively. [Conclusion] The azido-containing sugar unit could be incorporated into the structure of bacterial cell wall PG by the preincubation of bacteria with Ac3GlcNAz-1-P, allowing the PG of living bacterial to be easily labelled by alkyne-bearing fluorescent reagents (bioorthogonal fluorescent probes). This method would be useful for the study of biological and pathological functions of bacterial PG.

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袁雪,孙欢,丁超,程水红,傅立峰,李学兵. 大肠杆菌细胞壁肽聚糖的化学修饰及荧光标记[J]. 微生物学报, 2020, 60(1): 49-59

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  • 收稿日期:2019-03-04
  • 最后修改日期:2019-04-19
  • 在线发布日期: 2020-01-10
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