Whole‐Body Imaging of Infection Using Bioluminescence

Ying Kong1, Yanlin Shi1, Mihee Chang1, Ali R. Akin2, Kevin P. Francis2, Ning Zhang2, Tamara L. Troy2, Hequn Yao3, Jianghong Rao3, Suat L. G. Cirillo1, Jeffrey D. Cirillo1

1 Department of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, Texas, 2 Caliper Life Sciences, Alameda, California, 3 Molecular Imaging Program at Stanford, Departments of Radiology and Chemistry, Stanford University, Stanford, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2C.4
DOI:  10.1002/9780471729259.mc02c04s21
Online Posting Date:  May, 2011
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Abstract

Bioluminescence imaging is a powerful technique to visualize and monitor biological processes in numerous systems. This unit describes two strategies for bioluminescence imaging that can be used to study bacterial infection in mice. One method is to express a luciferase gene in the bacteria; the second method is to use bacteria that express both a luciferase and β‐lactamase along with a substrate containing caged luciferin, which is released by β‐lactamase hydrolysis and reacts with luciferase to generate light. For both strategies, bioluminescent signals are imaged using an IVIS live animal imaging system (Caliper Life Sciences). The bioluminescence images are analyzed to localize bioluminescent bacteria, quantify signal, and determine the wavelengths of the signals produced. The correlation of bacterial numbers with signal intensity in vivo can be determined, allowing a quantitative measure of bacterial numbers in mice in real time. Methods are described in detail to facilitate successful application of these emerging technologies in nearly any experimental system. Curr. Protoc. Microbiol. 21:2C.4.1‐2C.4.17. © 2011 by John Wiley & Sons, Inc.

Keywords: bioluminescence; fluorescence; mice; in vivo imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Imaging M. smegmatis Lung Infection in Live Mice Using Luciferase
  • Basic Protocol 2: Imaging M. bovis BCG Subcutaneous Infection in Live Mice Using Luciferase
  • Basic Protocol 3: Sequential Reporter Enzyme Luminescence Imaging of Mice with Bluco, a Caged Luciferin Substrate
  • Support Protocol 1: 3D Bioluminescent Imaging via DLIT Acquisition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Imaging M. smegmatis Lung Infection in Live Mice Using Luciferase

  Materials
  • Mycobacterium smegmatis strain expressing a Click Beetle Red luciferase gene (available from Dr. Cirillo's lab; )
  • Female nu/nu mice, 6 to 8 weeks of age
  • Isoflurane
  • 20 mg/ml luciferin in PBS (see recipe)
  • XGI‐8 gas anesthesia system (Caliper Life Sciences; http://www.caliperls.com/)
  • IVIS imaging system (Caliper Life Sciences; http://www.caliperls.com/)
  • Living Image software (Caliper Life Sciences; http://www.caliperls.com/)
  • Additional reagents and equipment for growing mycobacterial inoculum and mycobacterial infection of mouse lungs (unit 2.3, protocol 2), isoflurane anesthesia of mice (unit 2.3, Support Protocol 3), and 3D imaging ( protocol 4 in this unit)

Basic Protocol 2: Imaging M. bovis BCG Subcutaneous Infection in Live Mice Using Luciferase

  Materials
  • Cultures of M. bovis BCG strains expressing luciferase genes CBRluc and FFluc using plasmids derived from pJDC89 (Mehta et al., ) by inserting luciferase genes between NheI and PacI sites (available from Dr. Cirillo's lab; )
  • 2 mM luciferin solution (in 0.45 M sodium citrate, pH 5.0; see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Isoflurane
  • 20 mg/ml luciferin in PBS (see recipe)
  • White 96‐well microtiter plates
  • Microtiter plate reader with automatic injection function
  • Refrigerated centrifuge with microtiter plate carrier
  • Balance for weighing mice
  • XGI‐8 gas anesthesia system (Caliper Life Sciences; http://www.caliperls.com/)
  • Transparent nose cones (Caliper Life Sciences; http://www.caliperls.com)
  • IVIS imaging system (Caliper Life Sciences; http://www.caliperls.com/)
  • Light baffle divider (Caliper Life Sciences; http://www.caliperls.com)
  • Living Image software (Caliper Life Sciences; http://www.caliperls.com/)
  • Microsoft Excel software
  • Additional reagents and equipment for preparation of mycobacterial cultures for inoculation (unit 2.3, Support Protocol 1), infection of mice (unit 2.3, Support Protocol 2), isoflurane anesthesia of mice (unit 2.3, Support Protocol 3), and 3D imaging ( protocol 4 in this unit)

Basic Protocol 3: Sequential Reporter Enzyme Luminescence Imaging of Mice with Bluco, a Caged Luciferin Substrate

  Materials
  • M. smegmatis strain expressing Click Beetle Red (CBR) luciferase (available from Dr. Cirillo's lab; )
  • M‐OADC‐TW medium (unit 2.3) containing 80 µg/ml hygromycin
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4, containing 0.05% (v/v) Tween 80
  • Citrate buffer (see recipe)
  • 2.5 µg/ml Bluco solution (see recipe)
  • M‐OADC agar plate (unit 2.3) containing 80 µg/ml hygromycin
  • Mice
  • Spectrophotometer
  • White flat‐bottom 96‐well microtiter plate, sterile, with lid
  • Microtiter plate reader
  • Incubator
  • Clipper
  • Sterile needles and syringes
  • Mice
  • IVIS Imaging System (Caliper Life Sciences)
  • Additional reagents and equipment for preparing mycobacterial inoculum (unit 2.3, Support Protocol 1), infection of mice (unit 2.3, Support Protocol 2), acquisition and analysis of images ( protocol 2 in this unit), and 3D analysis ( protocol 4 in this unit)

Support Protocol 1: 3D Bioluminescent Imaging via DLIT Acquisition

  Materials
  • IVIS system
  • Living Image software
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Figures

Videos

Literature Cited

Literature Cited
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   Contag, C.H., Contag, P.R., Mullins, J.I., Spilman, S.D., Stevenson, D.K., and Benaron, D.A. 1995. Photonic detection of bacterial pathogens in living hosts. Mol. Microbiol. 18:593‐603.
   de Wet, J.R., Wood, K.V., Helinski, D.R., and DeLuca, M. 1985. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 82:7870‐7873.
   Francis, K.P., Joh, D., Bellinger‐Kawahara, C., Hawkinson, M.J., Purchio, T.F., and Contag, P.R. 2000. Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct. Infect. Immun. 68:3594‐3600.
   Francis, K.P., Yu, J., Bellinger‐Kawahara, C., Joh, D., Hawkinson, M.J., Xiao, G., Purchio, T.F., Caparon, M.G., Lipsitch, M., and Contag, P.R. 2001. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram‐positive lux transposon. Infect. Immun. 69:3350‐3358.
   Hastings, J.W., Weber, K., Friedland, J., Eberhard, A., Mitchell, G.W., and Gunsalus, A. 1969. Structurally distinct bacterial luciferases. Biochemistry 8:4681‐4689.
   Hutchens, M. and Luker, G.D. 2007. Applications of bioluminescence imaging to the study of infectious diseases. Cell. Microbiol. 9:2315‐2322.
   Lorenz, W.W., McCann, R.O., Longiaru, M., and Cormier, M.J. 1991. Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc. Natl. Acad. Sci. U.S.A. 88:4438‐4442.
   Mehta, P.K., Pandey, A.K., Subbian, S., El‐Etr, S.H., Cirillo, S.L. , Samrakandi, M.M., and Cirillo, J.D. 2006. Identification of Mycobacterium marinum macrophage infection mutants. Microb. Pathog. 40:139‐151.
   Meighen, E.A. 1991. Molecular biology of bacterial bioluminescence. Microbiol. Rev. 55:123‐142.
   Rice, B.W., Cable, M.D., and Nelson, M.B. 2001. In vivo imaging of light‐emitting probes. J. Biomed. Opt. 6:432‐440.
   Weissleder, R. 2001. A clearer vision for in vivo imaging. Nat. Biotechnol. 19:316‐317.
   Wilson, T. and Hastings, J.W. 1998. Bioluminescence. Annu. Rev. Cell. Dev. Biol. 14:197‐230.
   Wood, K.V., Lam, Y.A., and McElroy, W.D. 1989. Introduction to beetle luciferases and their applications. J. Biolumin. Chemilumin. 4:289‐301.
   Zhao, H., Doyle, T.C., Coquoz, O., Kalish, F., Rice, B.W., and Contag, C.H. 2005. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J. Biomed. Opt. 10:41210.
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