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Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis

Nature Chemical Biology volume 7, pages 228–235 (2011)Cite this article

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Abstract

The detection of tuberculosis currently relies upon insensitive and unspecific techniques; newer diagnostics would ideally co-opt specific bacterial processes to provide real-time readouts. The trehalose mycolyltransesterase enzymes (antigens 85A, 85B and 85C (Ag85A, Ag85B, Ag85C)) serve as essential mediators of cell envelope function and biogenesis in Mycobacterium tuberculosis. Through the construction of a systematically varied sugar library, we show here that Ag85 enzymes have exceptionally broad substrate specificity. This allowed exogenously added synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. Even bulky substituents, such as a fluorescein-containing trehalose probe (FITC-trehalose), were incorporated by growing bacilli, thereby producing fluorescent bacteria; microscopy revealed selective labeling of poles and membrane. Addition of FITC-trehalose to M. tuberculosis–infected macrophages allowed selective, sensitive detection of M. tuberculosis within infected mammalian macrophages. These studies suggest that analogs of trehalose may prove useful as probes of function and for other imaging modalities.

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Figure 1: Ag85 structure and enzyme activity.
Figure 2: Trehalose is incorporated into M. tuberculosis in vitro and in macrophages.
Figure 3: Trehalose library.
Figure 4: Labeling of individual bacilli in vitro and within infected macrophages.

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Acknowledgements

We thank T. Claridge, B. Odell and T. Jackson for assistance with NMR spectra and C. Sparrow, J. McCullagh and R. Procter for their assistance with mass spectrometry. O. Schwartz, L. Koo, M. Gastinger, S. Becker and J. Kabat provided tremendous assistance on all imaging of M. tuberculosis. We thank the Colorado State “Tuberculosis Vaccine Testing and Research Materials” Contract (Colorado State University, Fort Collins) and G.J. Davies (University of York) for providing Ag85 and OtsA plasmids, respectively. We thank A. Sher (US National Institute of Allergy and Infectious Disease, US National Institutes of Health) for M. bovis BCG expressing DSRed1 and T. Oh (Yonsei University) for H37Rv carrying the mCherry-PMV261 plasmid. This study was supported (in part) by the Intramural Research Program of the US National Institutes of Health, the US National Institute of Allergy and Infectious Disease (C.E.B.), the Rhodes Trust (K.M.B.), a Marie Curie Intra European Fellowship (O.B.), the Engineering and Physical Sciences Research Council (Platform Grant to B.G.D.) and the Bill and Melinda Gates Foundation through the Tuberculosis Drug Accelerator Program (C.E.B., B.G.D.).

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Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, UK

    Keriann M Backus, Conor S Barry, Omar Boutureira, Mitul K Patel, François D'Hooge, Seung Seo Lee & Benjamin G Davis

  2. Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, US National Institute of Allergy and Infectious Disease, Bethesda, Maryland, USA

    Keriann M Backus, Helena I Boshoff, Laura E Via, Kapil Tahlan & Clifton E Barry III

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  1. Keriann M Backus
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Contributions

K.M.B., H.I.B., C.S.B., L.E.V., C.E.B. and B.G.D. designed experiments. K.M.B., O.B., M.K.P., F.D. and S.S.L. synthesized compounds. K.M.B., K.T. and H.I.B. performed uptake experiments. C.S.B. and K.M.B. performed substrate screens. K.M.B., H.I.B., C.S.B., C.E.B. and B.G.D. analyzed results. K.M.B., C.E.B. and B.G.D. wrote the paper.

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Correspondence to Clifton E Barry III or Benjamin G Davis.

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The work from this paper has been filed as a patent application for the detection of mycobacteria by the University of Oxford and the US National Institutes of Health and, if licensed, will afford authors royalties in line with the set down guidelines of both institutions.

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Backus, K., Boshoff, H., Barry, C. et al. Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis. Nat Chem Biol 7, 228–235 (2011). https://doi.org/10.1038/nchembio.539

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