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. 2012 Oct 3;134(39):16123-6.
doi: 10.1021/ja3062419. Epub 2012 Sep 24.

Probing the mycobacterial trehalome with bioorthogonal chemistry

Benjamin M Swarts  1 , Cynthia M Holsclaw, John C Jewett, Marina Alber, Douglas M Fox, M Sloan Siegrist, Julie A Leary, Rainer Kalscheuer, Carolyn R Bertozzi
Affiliations
Free PMC article

Probing the mycobacterial trehalome with bioorthogonal chemistry

Benjamin M Swarts et al. J Am Chem Soc. .
Free PMC article

Abstract

Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome.

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Figures

Figure 1
Figure 1
(A) Common trehalose glycolipids in mycobacteria. (B) Synthetic TreAz analogues used in this study. (C) TreAz-based bioorthogonal chemical reporter strategy.
Figure 2
Figure 2
Trehalose metabolism in mycobacteria. Exogenous TreAz can label glycolipids via the Ag85 or recycling pathways. AG, arabinogalactan; CL, capsular layer; MM, mycomembrane; PG, peptidoglycan; PM, plasma membrane. Exact extracellular location of Ag85 is unknown.
Figure 3
Figure 3
(A) Flow cytometry analysis of TreAz-labeled Msmeg reacted with BARAC-Fluor. Error bars denote the standard deviation of three replicate experiments. (B) Fluorescence microscopy of TreAz-labeled Msmeg reacted with alk-AF488 via CuAAC. Scale bars, 5 μm.
Figure 4
Figure 4
(A) Flow cytometry analysis of TreAz-labeled Msmeg strains. Error bars denote the standard deviation from three replicate experiments. * p < 0.05. (B) HPAEC-PAD analysis of cytosolic extracts from TreAz-treated wild-type Msmeg. Dotted lines represent retention times for authentic standards (see SI for details).
Figure 5
Figure 5
Flow cytometry analysis of TreAz-labeled Mtb and BCG strains. (A) Wild-type Mtb H37Rv and BCG; (B) wild-type BCG, BCG ΔlpqY-sugC, and the corresponding complemented strain. Error bars denote the standard deviation from three replicate experiments.
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