Chemical modification and fluorescence labeling of Escherichia coli cell wall peptidoglycan

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.