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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

Bioluminescence levels in M. tuberculosis and M. smegmatis are comparable in vitro.Relative light units (RLUs) were measured in 10 M. smegmatis and 10 M. tuberculosis clones transformed with pMV306hsp+FFluc (a), pMV306hsp+Gluc (b) or pMV306hsp+Lux (c). Results are corrected for the background. Statistical significance was evaluated by the Mann-Whitney non-parametric test for Lux, and by unpaired t test for FFluc and Gluc (data normality passed) and those found to be significant (p<0.05) are indicated with *.
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pone-0010777-g004: Bioluminescence levels in M. tuberculosis and M. smegmatis are comparable in vitro.Relative light units (RLUs) were measured in 10 M. smegmatis and 10 M. tuberculosis clones transformed with pMV306hsp+FFluc (a), pMV306hsp+Gluc (b) or pMV306hsp+Lux (c). Results are corrected for the background. Statistical significance was evaluated by the Mann-Whitney non-parametric test for Lux, and by unpaired t test for FFluc and Gluc (data normality passed) and those found to be significant (p<0.05) are indicated with *.

Mentions: The three reporters were also tested in M. tuberculosis by electroporating pMV306hsp+FFluc, pMV306hsp+Gluc and pMV306hsp+Lux and measuring the bioluminescence of 10 randomly selected clones (Fig. 4). The signal obtained in M. tuberculosis was slightly lower than that of M. smegmatis for FFluc (1.1×107 and 1.5×107 RLUs, respectively), whereas no significant differences were detected for Gluc and Lux (5.8×105 vs. 8.4×105 RLUs, and 3.6×104 vs. 4.6×104 RLUs, correspondingly).


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)

Bioluminescence levels in M. tuberculosis and M. smegmatis are comparable in vitro.Relative light units (RLUs) were measured in 10 M. smegmatis and 10 M. tuberculosis clones transformed with pMV306hsp+FFluc (a), pMV306hsp+Gluc (b) or pMV306hsp+Lux (c). Results are corrected for the background. Statistical significance was evaluated by the Mann-Whitney non-parametric test for Lux, and by unpaired t test for FFluc and Gluc (data normality passed) and those found to be significant (p<0.05) are indicated with *.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010777-g004: Bioluminescence levels in M. tuberculosis and M. smegmatis are comparable in vitro.Relative light units (RLUs) were measured in 10 M. smegmatis and 10 M. tuberculosis clones transformed with pMV306hsp+FFluc (a), pMV306hsp+Gluc (b) or pMV306hsp+Lux (c). Results are corrected for the background. Statistical significance was evaluated by the Mann-Whitney non-parametric test for Lux, and by unpaired t test for FFluc and Gluc (data normality passed) and those found to be significant (p<0.05) are indicated with *.
Mentions: The three reporters were also tested in M. tuberculosis by electroporating pMV306hsp+FFluc, pMV306hsp+Gluc and pMV306hsp+Lux and measuring the bioluminescence of 10 randomly selected clones (Fig. 4). The signal obtained in M. tuberculosis was slightly lower than that of M. smegmatis for FFluc (1.1×107 and 1.5×107 RLUs, respectively), whereas no significant differences were detected for Gluc and Lux (5.8×105 vs. 8.4×105 RLUs, and 3.6×104 vs. 4.6×104 RLUs, correspondingly).

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