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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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Expression of luciferases using promoters Phsp60 and PG13 leads to greater light production.Luminescence of M. smegmatis expressing ffluc (a), gluc (b) and lux (c) was assayed using Phsp60, PmyctetO, and PG13 in the integrating vector pMV306. Each dot represents a randomly selected transformant. Results are given as relative light units (RLUs) and are corrected for the background. Statistical significance was evaluated by the Kruskal–Wallis test with subgroup analysis performed by Dunn's multiple comparison test and those found to be significant (p<0.05) are indicated with *.
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pone-0010777-g002: Expression of luciferases using promoters Phsp60 and PG13 leads to greater light production.Luminescence of M. smegmatis expressing ffluc (a), gluc (b) and lux (c) was assayed using Phsp60, PmyctetO, and PG13 in the integrating vector pMV306. Each dot represents a randomly selected transformant. Results are given as relative light units (RLUs) and are corrected for the background. Statistical significance was evaluated by the Kruskal–Wallis test with subgroup analysis performed by Dunn's multiple comparison test and those found to be significant (p<0.05) are indicated with *.

Mentions: Next, we tested different promoters to drive expression of the luciferase genes. Using the integrating vector pMV306, each reporter gene was cloned in front of previously described strong promoters: Phsp60, PmyctetO [42], and PG13 [43], [44]. These constructs were introduced into M. smegmatis, and the luminescence of 10 randomly selected transformants analysed (Fig. 2). Similar luminescence values were obtained among strains expressing either FFluc or Gluc under the control of either Phsp60 or PG13 (median values of 1.5×107 and 2.3×107 RLUs respectively for FFluc, 8.9×106 and 5.05×106 RLUs for Gluc), while production of light from PmyctetO clones was 3–13 times lower. In the case of Lux, the highest luminescence was achieved using Phsp60 (3.6×104 RLUs), followed by PG13 (9 times lower), and PmyctetO (180 times lower than that of Phsp60). Consequently Phsp60 was the promoter chosen for expression of the luminescent reporters. Additionally, in order to increase the amount of substrate synthesised by the Lux operon and in this way the amount of luminescence, PG13 was cloned in front of luxC in pMV306hsp+Lux. This resulted in a 6-fold increase in the luminescence activity compared to the original pMV306hsp+Lux, from 3.6×104 to 2.25×105 RLUs (Fig. 2C).


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)

Expression of luciferases using promoters Phsp60 and PG13 leads to greater light production.Luminescence of M. smegmatis expressing ffluc (a), gluc (b) and lux (c) was assayed using Phsp60, PmyctetO, and PG13 in the integrating vector pMV306. Each dot represents a randomly selected transformant. Results are given as relative light units (RLUs) and are corrected for the background. Statistical significance was evaluated by the Kruskal–Wallis test with subgroup analysis performed by Dunn's multiple comparison test 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-g002: Expression of luciferases using promoters Phsp60 and PG13 leads to greater light production.Luminescence of M. smegmatis expressing ffluc (a), gluc (b) and lux (c) was assayed using Phsp60, PmyctetO, and PG13 in the integrating vector pMV306. Each dot represents a randomly selected transformant. Results are given as relative light units (RLUs) and are corrected for the background. Statistical significance was evaluated by the Kruskal–Wallis test with subgroup analysis performed by Dunn's multiple comparison test and those found to be significant (p<0.05) are indicated with *.
Mentions: Next, we tested different promoters to drive expression of the luciferase genes. Using the integrating vector pMV306, each reporter gene was cloned in front of previously described strong promoters: Phsp60, PmyctetO [42], and PG13 [43], [44]. These constructs were introduced into M. smegmatis, and the luminescence of 10 randomly selected transformants analysed (Fig. 2). Similar luminescence values were obtained among strains expressing either FFluc or Gluc under the control of either Phsp60 or PG13 (median values of 1.5×107 and 2.3×107 RLUs respectively for FFluc, 8.9×106 and 5.05×106 RLUs for Gluc), while production of light from PmyctetO clones was 3–13 times lower. In the case of Lux, the highest luminescence was achieved using Phsp60 (3.6×104 RLUs), followed by PG13 (9 times lower), and PmyctetO (180 times lower than that of Phsp60). Consequently Phsp60 was the promoter chosen for expression of the luminescent reporters. Additionally, in order to increase the amount of substrate synthesised by the Lux operon and in this way the amount of luminescence, PG13 was cloned in front of luxC in pMV306hsp+Lux. This resulted in a 6-fold increase in the luminescence activity compared to the original pMV306hsp+Lux, from 3.6×104 to 2.25×105 RLUs (Fig. 2C).

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