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

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.

References

  1. 1

    Dye, C. et al. Measuring tuberculosis burden, trends, and the impact of control programmes. Lancet Infect. Dis. 8, 233–243 (2008).

    CAS  Article  Google Scholar 

  2. 2

    Dinnes, J. et al. A systematic review of rapid diagnostic tests for the detection of tuberculosis infection. Health Technol. Assess. 11, 1–196 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Ling, D. I., Flores, L. L., Riley, L. W. & Pai, M. Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression. PLoS One 3, e1536 (2008).

    Article  Google Scholar 

  4. 4

    Greco, S., Girardi, E., Navarra, A. & Saltini, C. Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis. Thorax 61, 783–790 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Boehme, C. C. et al. Rapid molecular detection of tuberculosis and rifampin resistance. N. Engl. J. Med. 363, 1005–1015 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Hughes, R., Wonderling, D., Li, B. & Higgins, B. The cost effectiveness of nucleic acid amplification techniques for the diagnosis of tuberculosis. Respir. Med. 106, 300–307 (2012).

    Article  Google Scholar 

  7. 7

    Flores, A. R., Parsons, L. M. & Pavelka, M. S. Jr Genetic analysis of the beta-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to beta-lactam antibiotics. Microbiology 151, 521–532 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Hugonnet, J. E., Tremblay, L. W., Boshoff, H. I., Barry, C. E. II & Blanchard, J. S. Meropenem–clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science 323, 1215–1218 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Boyd, D. B. & Lunn, W. H. Electronic structures of cephalosporins and penicillins. 9. Departure of a leaving group in cephalosporins. J. Med. Chem. 22, 778–784 (1979).

    CAS  Article  Google Scholar 

  10. 10

    Faraci, W. S. & Pratt, R. F. Elimination of a good leaving group from the 3′-position of a cephalosporin need not be concerted with beta-lactam ring-opening – TEM-2 beta-lactamase-catalyzed hydrolysis of pyridine-2-azo-4′-(N′,N′-dimethylaniline) cephalosporin (PADAC) and of cephaloridine. J. Am. Chem. Soc. 106, 1489–1490 (1984).

    CAS  Article  Google Scholar 

  11. 11

    Faraci, W. S. & Pratt, R. F. Mechanism of inhibition of the PC1 beta-lactamase of Staphylococcus aureus by cephalosporins: importance of the 3′-leaving group. Biochemistry 24, 903–910 (1985).

    CAS  Article  Google Scholar 

  12. 12

    Pratt, R. F. & Faraci, W. S. Direct observation by proton NMR of cephalosporoate intermediates in aqueous solution during the hydrazinolysis and beta-lactamase-catalyzed hydrolysis of cephalosporins with 3′ leaving groups: kinetics and equilibria of the 3′ elimination reaction. J. Am. Chem. Soc. 108, 5328–5333 (1986).

    CAS  Article  Google Scholar 

  13. 13

    Zlokarnik, G. et al. Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279, 84–88 (1998).

    CAS  Article  Google Scholar 

  14. 14

    Gao, W., Xing, B., Tsien, R. Y. & Rao, J. Novel fluorogenic substrates for imaging beta-lactamase gene expression. J. Am. Chem. Soc. 125, 11146–11147 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Xing, B., Khanamiryan, A. & Rao, J. Cell-permeable near-infrared fluorogenic substrates for imaging beta-lactamase activity. J. Am. Chem. Soc. 127, 4158–4159 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Yao, H., So, M. K. & Rao, J. A bioluminogenic substrate for in vivo imaging of beta-lactamase activity. Angew. Chem. Int. Ed. 46, 7031–7034 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Rukavishnikov, A., Gee, K. R., Johnson, I. & Corry, S. Fluorogenic cephalosporin substrates for beta-lactamase TEM-1. Anal. Biochem. 419, 9–16 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Kong, Y. et al. Imaging tuberculosis with endogenous beta-lactamase reporter enzyme fluorescence in live mice. Proc. Natl Acad. Sci. USA 107, 12239–12244 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Banerjee, S., Pieper, U., Kapadia, G., Pannell, L. K. & Herzberg, O. Role of the Omega-loop in the activity, substrate specificity, and structure of class A beta-lactamase. Biochemistry 37, 3286–3296 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Knox, J. R. Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases – mutations, specificity, and 3-dimensional structure. Antimicrob. Agents Chemother. 39, 2593–2601 (1995).

    CAS  Article  Google Scholar 

  21. 21

    Albrecht, H. A. et al. Cephalosporin 3′-quinolone esters with a dual mode of action. J. Med. Chem. 33, 77–86 (1990).

    CAS  Article  Google Scholar 

  22. 22

    Baldwin, J. E., Urban, F. J., Cooper, R. D. G. & Jose, F. L. Direct 6-methoxylation of penicillin derivatives – convenient pathway to substituted beta-lactam antibiotics. J. Am. Chem. Soc. 95, 2401–2403 (1973).

    CAS  Article  Google Scholar 

  23. 23

    Wang, F., Cassidy, C. & Sacchettini, J. C. Crystal structure and activity studies of the Mycobacterium tuberculosis beta-lactamase reveal its critical role in resistance to beta-lactam antibiotics. Antimicrob. Agents Chemother. 50, 2762–2771 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Minasov, G., Wang, X. J. & Shoichet, B. K. An ultrahigh resolution structure of TEM-1 beta-lactamase suggests a role for Glu166 as the general base in acylation. J. Am. Chem. Soc. 124, 5333–5340 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Urano, Y. et al. Evolution of fluorescein as a platform for finely tunable fluorescence probes. J. Am. Chem. Soc. 127, 4888–4894 (2005).

    CAS  Article  Google Scholar 

  26. 26

    McNerney, R. & Daley, P. Towards a point-of-care test for active tuberculosis: obstacles and opportunities. Nature Rev. Microbiol. 9, 204–213 (2011).

    CAS  Article  Google Scholar 

  27. 27

    Backus, K. M., et al. Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis. Nature Chem. Biol. 7, 228–235 (2011).

    CAS  Article  Google Scholar 

  28. 28

    Ioerger, T. R., et al. The non-clonality of drug resistance in Beijing-genotype isolates of Mycobacterium tuberculosis from the Western Cape of South Africa. BMC Genomics 11, 670 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Kwon, H. H., Tomioka, H. & Saito, H. Distribution and characterization of beta-lactamases of mycobacteria and related organisms. Tuber. Lung Dis. 76, 141–148 (1995).

    CAS  Article  Google Scholar 

  30. 30

    Majiduddin, F. K., Materon, I. C. & Palzkill, T. G. Molecular analysis of beta-lactamase structure and function. Int. J. Med. Microbiol. 292, 127–137 (2002).

    CAS  Article  Google Scholar 

  31. 31

    Petrosino, J., Cantu, C. III & Palzkill, T. β-Lactamases: protein evolution in real time. Trends Microbiol. 6, 323–327 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Hugonnet, J. E. & Blanchard, J. S. Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate. Biochemistry 46, 11998–12004 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Tremblay, L. W., Fan, F. & Blanchard, J. S. Biochemical and structural characterization of Mycobacterium tuberculosis beta-lactamase with the carbapenems ertapenem and doripenem. Biochemistry 49, 3766–3773 (2010).

    CAS  Article  Google Scholar 

  34. 34

    Mitchell, R. S., Kumar, V., Robbins, S. L., Abbas, A. K. & Fausto, N. Robbins Basic Pathology (Saunders/Elsevier, 2007).

    Google Scholar 

  35. 35

    McPherson, A. Preparation and Analysis of Protein Crystals (Waverly, 1982).

    Google Scholar 

  36. 36

    Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).

    Article  Google Scholar 

  37. 37

    Mccoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  38. 38

    Bailey, S. The CCP4 suite – programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

    Article  Google Scholar 

  39. 39

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

Download references

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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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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