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Optimisation of bioluminescent reporters for use with mycobacteria.

Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible U, Robertson BD, Wiles S - PLoS ONE (2010)

Bottom Line: We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence.While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo.Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Imperial College London, London, UK.

ABSTRACT

Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.

Methodology/principal findings: One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.

Conclusions/significance: While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

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Related in: MedlinePlus

BLI of ffluc-expressing M. tuberculosis.Mice were inoculated endotracheally with 5×106 CFU of either wild-type M. tuberculosis (control) or FFluc-producing M. tuberculosis. 20 µl of 30 mg ml−1 luciferin was administered intranasally and mice were imaged 5–10 min after. Mice were contained in a large air-tight box for safety considerations. The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image of peak bioluminescence (given as photons s−1 cm−2 sr−1). Red represents the most intense light emission while blue correspond to the weakest signal. The colour bar indicates relative signal intensity. Mice were imaged with an integration time of 1 min.
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pone-0010777-g014: BLI of ffluc-expressing M. tuberculosis.Mice were inoculated endotracheally with 5×106 CFU of either wild-type M. tuberculosis (control) or FFluc-producing M. tuberculosis. 20 µl of 30 mg ml−1 luciferin was administered intranasally and mice were imaged 5–10 min after. Mice were contained in a large air-tight box for safety considerations. The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image of peak bioluminescence (given as photons s−1 cm−2 sr−1). Red represents the most intense light emission while blue correspond to the weakest signal. The colour bar indicates relative signal intensity. Mice were imaged with an integration time of 1 min.

Mentions: Finally, we also imaged mice infected with ffluc-expressing M. tuberculosis after administering intranasal luciferin. Bioluminescence was detected in the lungs of mice receiving ffluc-expressing bacteria, while no signal was observed in the control mouse infected with the wild-type M. tuberculosis H37Rv (Fig. 14).


Optimisation of bioluminescent reporters for use with mycobacteria.

Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible U, Robertson BD, Wiles S - PLoS ONE (2010)

BLI of ffluc-expressing M. tuberculosis.Mice were inoculated endotracheally with 5×106 CFU of either wild-type M. tuberculosis (control) or FFluc-producing M. tuberculosis. 20 µl of 30 mg ml−1 luciferin was administered intranasally and mice were imaged 5–10 min after. Mice were contained in a large air-tight box for safety considerations. The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image of peak bioluminescence (given as photons s−1 cm−2 sr−1). Red represents the most intense light emission while blue correspond to the weakest signal. The colour bar indicates relative signal intensity. Mice were imaged with an integration time of 1 min.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875389&req=5

pone-0010777-g014: BLI of ffluc-expressing M. tuberculosis.Mice were inoculated endotracheally with 5×106 CFU of either wild-type M. tuberculosis (control) or FFluc-producing M. tuberculosis. 20 µl of 30 mg ml−1 luciferin was administered intranasally and mice were imaged 5–10 min after. Mice were contained in a large air-tight box for safety considerations. The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image of peak bioluminescence (given as photons s−1 cm−2 sr−1). Red represents the most intense light emission while blue correspond to the weakest signal. The colour bar indicates relative signal intensity. Mice were imaged with an integration time of 1 min.
Mentions: Finally, we also imaged mice infected with ffluc-expressing M. tuberculosis after administering intranasal luciferin. Bioluminescence was detected in the lungs of mice receiving ffluc-expressing bacteria, while no signal was observed in the control mouse infected with the wild-type M. tuberculosis H37Rv (Fig. 14).

Bottom Line: We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence.While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo.Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Imperial College London, London, UK.

ABSTRACT

Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.

Methodology/principal findings: One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.

Conclusions/significance: While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

Show MeSH
Related in: MedlinePlus