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Noninvasive imaging of Staphylococcus aureus infections with a nuclease-activated probe

Nature Medicine volume 20, pages 301–306 (2014)Cite this article

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Abstract

Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease (MN). Several short synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications and flanked with a fluorophore and quencher, were activated upon degradation by purified MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2′-O-methyl–modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This new bacterial imaging approach has potential clinical applicability for infections with S. aureus and several other medically important pathogens.

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Figure 1: Nucleic acid probe activation by MN and serum nucleases.
Figure 2: Activation of the Cy5. 5-TT probe by MN in vitro and in S. aureus–infected mice.
Figure 3: Nucleic acid probe activation in cultures of additional bacterial pathogens.

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References

  1. Agarwal, V., Chauhan, S. & Gupta, R.K. Pyomyositis. Neuroimaging Clin. N. Am. 21, 975–983 (2011).

    Article  Google Scholar 

  2. Carek, P.J., Dickerson, L.M. & Sack, J.L. Diagnosis and management of osteomyelitis. Am. Fam. Physician 63, 2413–2420 (2001).

    CAS  PubMed  Google Scholar 

  3. Clerc, O., Prod′hom, G., Greub, G., Zanetti, G. & Senn, L. Adult native septic arthritis: a review of 10 years of experience and lessons for empirical antibiotic therapy. J. Antimicrob. Chemother. 66, 1168–1173 (2011).

    Article  CAS  Google Scholar 

  4. Hatzenbuehler, J. & Pulling, T.J. Diagnosis and management of osteomyelitis. Am. Fam. Physician 84, 1027–1033 (2011).

    PubMed  Google Scholar 

  5. Hope-Roberts, M., Wainwright, M. & Horobin, R.W. Real-time imaging of bacteria in living mice using a fluorescent dye. Biotech. Histochem. 86, 104–107 (2011).

    Article  CAS  Google Scholar 

  6. Leevy, W.M. et al. Optical imaging of bacterial infection in living mice using a fluorescent near-infrared molecular probe. J. Am. Chem. Soc. 128, 16476–16477 (2006).

    Article  CAS  Google Scholar 

  7. Leevy, W.M. et al. Noninvasive optical imaging of Staphylococcus aureus bacterial infection in living mice using a bis-dipicolylamine-Zinc(ii) affinity group conjugated to a near-infrared fluorophore. Bioconjug. Chem. 19, 686–692 (2008).

    Article  CAS  Google Scholar 

  8. Ning, X. et al. Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity. Nat. Mater. 10, 602–607 (2011).

    Article  CAS  Google Scholar 

  9. Panizzi, P. et al. In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation. Nat. Med. 17, 1142–1146 (2011).

    Article  CAS  Google Scholar 

  10. White, A.G. et al. Optical imaging of bacterial infection in living mice using deep-red fluorescent squaraine rotaxane probes. Bioconjug. Chem. 21, 1297–1304 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Yang, W. Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 44, 1–93 (2011).

    Article  Google Scholar 

  13. Kelemen, B.R. et al. Hypersensitive substrate for ribonucleases. Nucleic Acids Res. 27, 3696–3701 (1999).

    Article  CAS  Google Scholar 

  14. Elias, D.R., Thorek, D.L., Chen, A.K., Czupryna, J. & Tsourkas, A. In vivo imaging of cancer biomarkers using activatable molecular probes. Cancer Biomark. 4, 287–305 (2008).

    Article  CAS  Google Scholar 

  15. Hernandez, F.J. et al. Degradation of nuclease-stabilized RNA oligonucleotides in Mycoplasma-contaminated cell culture media. Nucleic Acid Ther. 22, 58–68 (2012).

    Article  CAS  Google Scholar 

  16. Arnone, A. et al. The extracellular nuclease of Staphylococcus aureus: structures of the native enzyme and an enzyme-inhibitor complex at 4 A resolution. Proc. Natl. Acad. Sci. USA 64, 420–427 (1969).

    Article  CAS  Google Scholar 

  17. Cuatrecasas, P., Fuchs, S. & Anfinsen, C.B. Catalytic properties and specificity of the extracellular nuclease of Staphylococcus aureus. J. Biol. Chem. 242, 1541–1547 (1967).

    CAS  PubMed  Google Scholar 

  18. Cunningham, L., Catlin, B.W. & Privatdegarilhe, M. A deoxyribonuclease of Micrococcus pyogenes. J. Am. Chem. Soc. 78, 4642–4645 (1956).

    Article  CAS  Google Scholar 

  19. Boerlin, P., Kuhnert, P., Hussy, D. & Schaellibaum, M. Methods for identification of Staphylococcus aureus isolates in cases of bovine mastitis. J. Clin. Microbiol. 41, 767–771 (2003).

    Article  CAS  Google Scholar 

  20. Behlke, M.A. Chemical modification of siRNAs for in vivo use. Oligonucleotides 18, 305–319 (2008).

    Article  CAS  Google Scholar 

  21. Vester, B. & Wengel, J. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233–13241 (2004).

    Article  CAS  Google Scholar 

  22. Pochon, F. & Privat De Garilhe, M. Bull. Soc. Chim. Biol. (Paris) [Structure of oligonucleotides liberated by the action of desoxyribonuclease on Staphylococcus pyogenes] 42, 795–815 (1960).

    CAS  Google Scholar 

  23. Kiedrowski, M.R. et al. Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus. PLoS ONE 6, e26714 (2011).

    Article  CAS  Google Scholar 

  24. Beenken, K.E. et al. Epistatic relationships between sarA and agr in Staphylococcus aureus biofilm formation. PLoS ONE 5, e10790 (2010).

    Article  Google Scholar 

  25. Weissleder, R. & Ntziachristos, V. Shedding light onto live molecular targets. Nat. Med. 9, 123–128 (2003).

    Article  CAS  Google Scholar 

  26. Contag, C.H. et al. Photonic detection of bacterial pathogens in living hosts. Mol. Microbiol. 18, 593–603 (1995).

    Article  CAS  Google Scholar 

  27. Francis, K.P. et al. Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct. Infect. Immun. 68, 3594–3600 (2000).

    Article  CAS  Google Scholar 

  28. Xiong, Y.Q. et al. Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model. Antimicrob. Agents Chemother. 49, 380–387 (2005).

    Article  CAS  Google Scholar 

  29. Beenken, K.E., Spencer, H., Griffin, L.M. & Smeltzer, M.S. Impact of extracellular nuclease production on the biofilm phenotype of Staphylococcus aureus under in vitro and in vivo conditions. Infect. Immun. 80, 1634–1638 (2012).

    Article  CAS  Google Scholar 

  30. de Marie, S. Difficult-to-treat infections. Intensive Care Med. 16 (suppl. 3), S239–S242 (1990).

    Article  Google Scholar 

  31. Wu, P.I. & Edelman, E.R. Structural biomechanics modulate intramuscular distribution of locally delivered drugs. J. Biomech. 41, 2884–2891 (2008).

    Article  Google Scholar 

  32. Ferreira, B.T., Benchetrit, L.C., De Castro, A.C., Batista, T.G. & Barrucand, L. Extracellular deoxyribonucleases of streptococci: a comparison of their occurrence and levels of production among β-hemolytic strains of various serological groups. Zentralbl. Bakteriol. 277, 493–503 (1992).

    Article  CAS  Google Scholar 

  33. Ferrieri, P., Gray, E.D. & Wannamaker, L.W. Biochemical and immunological characterization of the extracellular nucleases of group B streptococci. J. Exp. Med. 151, 56–68 (1980).

    Article  CAS  Google Scholar 

  34. Moon, A.F. et al. Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae. Nucleic Acids Res. 39, 2943–2953 (2011).

    Article  CAS  Google Scholar 

  35. Marshall, M.V., Draney, D., Sevick-Muraca, E.M. & Olive, D.M. Single-dose intravenous toxicity study of IRDye 800CW in Sprague-Dawley rats. Mol. Imaging Biol. 12, 583–594 (2010).

    Article  Google Scholar 

  36. Kobayashi, H., Longmire, M.R., Ogawa, M. & Choyke, P.L. Rational chemical design of the next generation of molecular imaging probes based on physics and biology: mixing modalities, colors and signals. Chem. Soc. Rev. 40, 4626–4648 (2011).

    Article  CAS  Google Scholar 

  37. Matsui, A. et al. Real-time, near-infrared, fluorescence-guided identification of the ureters using methylene blue. Surgery 148, 78–86 (2010).

    Article  Google Scholar 

  38. Rasmussen, J.C. et al. Human lymphatic architecture and dynamic transport imaged using near-infrared fluorescence. Transl. Oncol. 3, 362–372 (2010).

    Article  Google Scholar 

  39. Sevick-Muraca, E.M. Translation of near-infrared fluorescence imaging technologies: emerging clinical applications. Annu. Rev. Med. 63, 217–231 (2012).

    Article  CAS  Google Scholar 

  40. Novick, R.P. Genetic systems in staphylococci. Methods Enzymol. 204, 587–636 (1991).

    Article  CAS  Google Scholar 

  41. Stoltz, D.A. et al. Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth. Sci. Transl. Med. 2, 29ra31 (2010).

    Article  Google Scholar 

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Acknowledgements

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health under award numbers AI083211 (A.R.H.) and AI101391 (J.O.M. II). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health. F.J.H. was supported by a postdoctoral fellowship from the American Heart Association. The authors thank J. Kavanaugh for technical assistance with bacterial cultures and R. Schmidt and J. Widness for sharing a Sysmex XT-200i Automated Hematology Analyzer.

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Authors and Affiliations

  1. Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Frank J Hernandez, Luiza I Hernandez & James O McNamara II

  2. Integrated DNA Technologies (IDT), Coralville, Iowa, USA

    Lingyan Huang, Kristy M Powers & Mark A Behlke

  3. Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Michael E Olson & Alexander R Horswill

  4. Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    David K Meyerholz

  5. Department of Radiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Daniel R Thedens

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Contributions

F.J.H. developed the experimental methodology, designed experiments, carried out experiments and analyzed data. L.H., M.E.O. and K.M.P. generated new reagents and provided crucial technical input. M.E.O. also measured the CFUs of bacterial cultures. L.I.H. measured blood counts of mice. D.K.M. carried out the histological staining and data analysis. D.R.T. carried out the magnetic resonance imaging and analysis. M.A.B. provided crucial input for probe design and generation, oversaw probe generation and contributed to the conceptual development of the project. J.O.M. II, A.R.H. and F.J.H. conceived of and developed the underlying concepts of the project. A.R.H. oversaw the generation of genetically modified bacteria and the preparation of bacterial cultures. J.O.M. II designed experiments, analyzed data, coordinated the various aspects of the project and wrote the manuscript with help from all of the authors.

Corresponding author

Correspondence to James O McNamara II.

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Competing interests

F.J.H., L.H., M.A.B., A.R.H. and J.O.M. II are inventors on a patent application submitted to the United States Patent and Trademark Office that covers the detection of bacterial nucleases with oligonucleotide probes. M.A.B., L.H. and K.M.P. are employed by Integrated DNA Technologies (IDT), which offers oligonucleotides for sale similar to some of the compounds described in the manuscript. IDT is not a publicly traded company, and these authors do not personally own any shares or equity in IDT.

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Hernandez, F., Huang, L., Olson, M. et al. Noninvasive imaging of Staphylococcus aureus infections with a nuclease-activated probe. Nat Med 20, 301–306 (2014). https://doi.org/10.1038/nm.3460

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