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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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Gaussia luciferase is secreted from mycobacterial cells.Luminescence (given as relative light units [RLUs]) was measured in culture, supernatant and cell samples of M. smegmatis producing Gluc Mycobacterium optimised with (GlucSS) or without (Gluc) signal peptide, Gluc wild-type with (GlucWT+SS) or without (GlucWT-SS) signal peptide, and FFluc as control. Assays were performed with three independent cultures and each culture was measured in duplicate. As the data was not normally distributed, median values are displayed (bar) with inter-quartile ranges (box), and highest and lowest values (whiskers).
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pone-0010777-g009: Gaussia luciferase is secreted from mycobacterial cells.Luminescence (given as relative light units [RLUs]) was measured in culture, supernatant and cell samples of M. smegmatis producing Gluc Mycobacterium optimised with (GlucSS) or without (Gluc) signal peptide, Gluc wild-type with (GlucWT+SS) or without (GlucWT-SS) signal peptide, and FFluc as control. Assays were performed with three independent cultures and each culture was measured in duplicate. As the data was not normally distributed, median values are displayed (bar) with inter-quartile ranges (box), and highest and lowest values (whiskers).

Mentions: It has previously been stated that Gluc is secreted from eukaryotic cells because of a signal peptide situated at the N-terminal end of the protein [9]. To determine if Gluc was also secreted in M. smegmatis, we examined the luminescence produced by the whole culture, the cells or the culture supernatant. Moreover, as a way to assess the role of the signal peptide, experiments were also performed using strains expressing codon-optimised and wild-type forms of Gluc without the signal sequence. As shown in Fig. 9, almost 100% of the luminescence was detected in the supernatant of all gluc-expressing M. smegmatis regardless of the presence or absence of the signal peptide and the codon usage of the gene used. In contrast, only 2% of the total luminescence was found in the supernatant of FFluc-producing M. smegmatis when analysed in a similar way. Surprisingly, the light output from Gluc was higher when the signal peptide was deleted (Fig. 9), both for the Mycobacterium optimised (8.2×105±5.6×104 RLUs compared to 1.75×105±3.8×104 RLUs with signal peptide) and the wild-type genes (1.5×105±1.36×104 RLUs compared to 2.1×104±1.3×103 RLUs).


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)

Gaussia luciferase is secreted from mycobacterial cells.Luminescence (given as relative light units [RLUs]) was measured in culture, supernatant and cell samples of M. smegmatis producing Gluc Mycobacterium optimised with (GlucSS) or without (Gluc) signal peptide, Gluc wild-type with (GlucWT+SS) or without (GlucWT-SS) signal peptide, and FFluc as control. Assays were performed with three independent cultures and each culture was measured in duplicate. As the data was not normally distributed, median values are displayed (bar) with inter-quartile ranges (box), and highest and lowest values (whiskers).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010777-g009: Gaussia luciferase is secreted from mycobacterial cells.Luminescence (given as relative light units [RLUs]) was measured in culture, supernatant and cell samples of M. smegmatis producing Gluc Mycobacterium optimised with (GlucSS) or without (Gluc) signal peptide, Gluc wild-type with (GlucWT+SS) or without (GlucWT-SS) signal peptide, and FFluc as control. Assays were performed with three independent cultures and each culture was measured in duplicate. As the data was not normally distributed, median values are displayed (bar) with inter-quartile ranges (box), and highest and lowest values (whiskers).
Mentions: It has previously been stated that Gluc is secreted from eukaryotic cells because of a signal peptide situated at the N-terminal end of the protein [9]. To determine if Gluc was also secreted in M. smegmatis, we examined the luminescence produced by the whole culture, the cells or the culture supernatant. Moreover, as a way to assess the role of the signal peptide, experiments were also performed using strains expressing codon-optimised and wild-type forms of Gluc without the signal sequence. As shown in Fig. 9, almost 100% of the luminescence was detected in the supernatant of all gluc-expressing M. smegmatis regardless of the presence or absence of the signal peptide and the codon usage of the gene used. In contrast, only 2% of the total luminescence was found in the supernatant of FFluc-producing M. smegmatis when analysed in a similar way. Surprisingly, the light output from Gluc was higher when the signal peptide was deleted (Fig. 9), both for the Mycobacterium optimised (8.2×105±5.6×104 RLUs compared to 1.75×105±3.8×104 RLUs with signal peptide) and the wild-type genes (1.5×105±1.36×104 RLUs compared to 2.1×104±1.3×103 RLUs).

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