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

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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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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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.

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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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