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Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe

Nature Chemistry volume 4, pages 802–809 (2012)Cite this article

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Abstract

Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100–200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens.

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Figure 1
Figure 2: Kinetic comparison of CDC probes with β-lactamases.
Figure 3: Comparison of BlaC and TEM-1 Bla active sites and substrate-specificity loops.
Figure 4: Active-site details of the BlaC-CDC-OMe (top) and BlaC–CDC-1 (bottom) acyl intermediate complexes.
Figure 5: β-Lactamase selectivity of green fluorescent probes CDG-1 and CDG-OMe.
Figure 6: Sensitivity and specificity of CDG-OMe in raw, unprocessed human sputum.

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Acknowledgements

This work was supported by grant 48523 from the Bill and Melinda Gates Foundation, the Welch Foundation grant no. A-0015 and NIH P01 68135 for TB Structural Genomics. We thank Bob Fader in the Scott & White Memorial Hospital (Temple, Texas) for providing sputum samples from cystic fibrosis patients.

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Author notes

  1. Hexin Xie, Joseph Mire and Ying Kong: These authors contributed equally to this work

Authors and Affiliations

  1. Departments of Radiology and Chemistry, Molecular Imaging Program at Stanford, Stanford University, 1201 Welch Road, Stanford, 94305-5484, California, USA

    Hexin Xie & Jianghong Rao

  2. Departments of Chemistry and Biochemistry and Biophysics, Texas A&M University, College Station, 77843, Texas, USA

    Joseph Mire & James C. Sacchettini

  3. Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Bryan, 77807, Texas, USA

    Ying Kong, MiHee Chang, Hany A. Hassounah & Jeffrey D. Cirillo

  4. Global BioDiagnostics Corp., 19 N. Main St, Temple, 76501, Texas, USA

    Chris N. Thornton

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Contributions

H.X. performed all the compound syntheses and characterizations, collected enzymatic kinetics and carried out the E. coli imaging. J.M. performed the crystallization and structural studies and analysed the data. Y.K., M.H.C. and H.A.H. performed the testing with BCG in human sputum. C.N.T. contributed the imaging box used for cellular phone imaging. H.X., J.M., Y.K., J.C.S., J.D.C. and J.R. conceived and designed the experiments. All authors discussed the results and commented on the manuscript. H.X., J.M., Y.K., J.C.S., J.D.C. and J.R. co-wrote the paper.

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Correspondence to Jianghong Rao.

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Xie, H., Mire, J., Kong, Y. et al. Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe. Nature Chem 4, 802–809 (2012). https://doi.org/10.1038/nchem.1435

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