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Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging.

ABSTRACT: For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH₂-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate.

SUBMITTER: Zambito G

PROVIDER: S-EPMC7811125 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging.

Zambito Giorgia G Hall Mary P MP Wood Monika G MG Gaspar Natasa N Ridwan Yanto Y Stellari Fabio F FF Shi Ce C Kirkland Thomas A TA Encell Lance P LP Löwik Clemens C Mezzanotte Laura L

iScience 20201226 1

For <i>in vivo</i> multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH<sub>2</sub>-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in ...[more]

PMID: 33490896

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Project description:BackgroundHuman African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.Methodology/principal findingsHere, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5'-variant surface glycoprotein untranslated region (UTR) and a downstream 3'-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.Conclusions/significanceThe optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.

Dataset Information

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging.

Publications

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging.

Similar Datasets

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