Imaging tuberculosis with endogenous -lactamase reporter enzyme fluorescence in live mice

Department of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, TX 77843, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2010; 107(27):12239-44. DOI: 10.1073/pnas.1000643107
Source: PubMed


The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for beta-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 +/- 2 x 10(2) colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 10(4) CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus Calmette-Guérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.

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    • "In-vivo fluorescent images were acquired using the In-Vivo Imaging System (IVIS) Spectrum (Caliper Corporation, Alameda, CA, USA). Images were acquired at each excitation and wavelength pair as previously described [61]. The fluorescent signal was obtained at a 535 nm and 605 nm excitation light and read at 580, 620, 640, 660, 680, 700 nm. "
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    ABSTRACT: Mycobacterium tuberculosis (M.tb) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/γc (null) mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34(+) fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45(+)) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-γ, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2-8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB.
    PLoS ONE 05/2013; 8(5):e63331. DOI:10.1371/journal.pone.0063331 · 3.23 Impact Factor
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    • "A different approach for in vivo imaging of mycobacteria was described in 2010 by Kong et al.27 In this case a fluorogenic substrate for the mycobacterial β-lactamase was used. "
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    Journal of Antimicrobial Chemotherapy 04/2013; 68(9). DOI:10.1093/jac/dkt155 · 5.31 Impact Factor
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    • "More recently, the same group developed a new method where fluorescence emitted from a reporter enzyme reaction is measured. Using this method, they were able to detect as few as 104 cfu BCG and 106 cfu Mtb in the lungs of living mice when substrate was given immediately after infection.29 The authors also report a significant difference in fluorescent signal after 6days of treatment with isoniazid and rifampicin in mice infected with 106 cfu Mtb. "
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    Journal of Antimicrobial Chemotherapy 05/2012; 67(8):1948-60. DOI:10.1093/jac/dks161 · 5.31 Impact Factor
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