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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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Bioluminescence correlates with cell density during exponential growth in vitro.Cultures of M. smegmatis pMVhsp+FFluc (a), pMVhsp+Gluc (b) and pMVhsp+Lux (c) were inoculated to an optical density (OD) at 600 nm of 0.1 and the OD and the luminescence [given as relative light units (RLUs)] measured over 28 h. The luminescence was measured with integration times of 5, 0.1, and 10 s respectively, and substrate concentrations of 470 µM luciferin for FFluc and 40 µM coelenterazine for Gluc. The values represented correspond to the means of two independent cultures measured in triplicate. The error bars indicate standard deviations. A near linear relationship was found between bioluminescence [given as RLUs] and colony counts (given as colony forming units [CFU]) for mid-log cultures of M. smegmatis pMVhsp+FFluc (d), pMVhsp+Gluc (e) and pMVhsp+Lux (f) using a plate luminometer.
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pone-0010777-g008: Bioluminescence correlates with cell density during exponential growth in vitro.Cultures of M. smegmatis pMVhsp+FFluc (a), pMVhsp+Gluc (b) and pMVhsp+Lux (c) were inoculated to an optical density (OD) at 600 nm of 0.1 and the OD and the luminescence [given as relative light units (RLUs)] measured over 28 h. The luminescence was measured with integration times of 5, 0.1, and 10 s respectively, and substrate concentrations of 470 µM luciferin for FFluc and 40 µM coelenterazine for Gluc. The values represented correspond to the means of two independent cultures measured in triplicate. The error bars indicate standard deviations. A near linear relationship was found between bioluminescence [given as RLUs] and colony counts (given as colony forming units [CFU]) for mid-log cultures of M. smegmatis pMVhsp+FFluc (d), pMVhsp+Gluc (e) and pMVhsp+Lux (f) using a plate luminometer.

Mentions: We then analysed luminescence production during the growth of the same luciferase-producing strains (Fig. 8). The level of luminescence correlated well with cell density during the exponential growth phase for all the three reporters. However, the signal dropped when the cultures of lux- and ffluc-expressing cells entered stationary phase with a total loss of 83% and 77% respectively between the time points 12 and 28 h (Fig. 8A, C). This is most likely related to a decrease in the metabolic activity of the bacteria, and therefore in the availability of the FMNH2 and ATP needed by Lux and FFluc, respectively. In contrast, luminescence from cells expressing the Gaussia luciferase, which does not require any bacterial cofactors to catalyse the luminescence reaction, remained related to cell numbers until the end of the growth curve (Fig. 8B). Moreover, using mid-log cultures a good correlation (Spearman r = 1) was found between colony forming units (CFU) and luminescence (measured with the plate luminometer) over a range of cell numbers: 104 and 108 CFU for ffluc-expressing M. smegmatis, 103 and 108 for lux-expressing cells, and 104–106 for Gluc producing M. smegmatis (Fig. 8D–F).


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 correlates with cell density during exponential growth in vitro.Cultures of M. smegmatis pMVhsp+FFluc (a), pMVhsp+Gluc (b) and pMVhsp+Lux (c) were inoculated to an optical density (OD) at 600 nm of 0.1 and the OD and the luminescence [given as relative light units (RLUs)] measured over 28 h. The luminescence was measured with integration times of 5, 0.1, and 10 s respectively, and substrate concentrations of 470 µM luciferin for FFluc and 40 µM coelenterazine for Gluc. The values represented correspond to the means of two independent cultures measured in triplicate. The error bars indicate standard deviations. A near linear relationship was found between bioluminescence [given as RLUs] and colony counts (given as colony forming units [CFU]) for mid-log cultures of M. smegmatis pMVhsp+FFluc (d), pMVhsp+Gluc (e) and pMVhsp+Lux (f) using a plate luminometer.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010777-g008: Bioluminescence correlates with cell density during exponential growth in vitro.Cultures of M. smegmatis pMVhsp+FFluc (a), pMVhsp+Gluc (b) and pMVhsp+Lux (c) were inoculated to an optical density (OD) at 600 nm of 0.1 and the OD and the luminescence [given as relative light units (RLUs)] measured over 28 h. The luminescence was measured with integration times of 5, 0.1, and 10 s respectively, and substrate concentrations of 470 µM luciferin for FFluc and 40 µM coelenterazine for Gluc. The values represented correspond to the means of two independent cultures measured in triplicate. The error bars indicate standard deviations. A near linear relationship was found between bioluminescence [given as RLUs] and colony counts (given as colony forming units [CFU]) for mid-log cultures of M. smegmatis pMVhsp+FFluc (d), pMVhsp+Gluc (e) and pMVhsp+Lux (f) using a plate luminometer.
Mentions: We then analysed luminescence production during the growth of the same luciferase-producing strains (Fig. 8). The level of luminescence correlated well with cell density during the exponential growth phase for all the three reporters. However, the signal dropped when the cultures of lux- and ffluc-expressing cells entered stationary phase with a total loss of 83% and 77% respectively between the time points 12 and 28 h (Fig. 8A, C). This is most likely related to a decrease in the metabolic activity of the bacteria, and therefore in the availability of the FMNH2 and ATP needed by Lux and FFluc, respectively. In contrast, luminescence from cells expressing the Gaussia luciferase, which does not require any bacterial cofactors to catalyse the luminescence reaction, remained related to cell numbers until the end of the growth curve (Fig. 8B). Moreover, using mid-log cultures a good correlation (Spearman r = 1) was found between colony forming units (CFU) and luminescence (measured with the plate luminometer) over a range of cell numbers: 104 and 108 CFU for ffluc-expressing M. smegmatis, 103 and 108 for lux-expressing cells, and 104–106 for Gluc producing M. smegmatis (Fig. 8D–F).

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