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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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Signal:noise for FFluc and Lux increases with integration time, and decreases for Gluc.Luminescence was measured using six different integration times and four different substrate concentrations for M. smegmatis producing FFluc (a) and Gluc (b), or without substrate for Lux (c). The background luminescence was obtained from cultures of M. smegmatis with the empty pMV306hsp. Assays were performed with three independent mid-log 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-g005: Signal:noise for FFluc and Lux increases with integration time, and decreases for Gluc.Luminescence was measured using six different integration times and four different substrate concentrations for M. smegmatis producing FFluc (a) and Gluc (b), or without substrate for Lux (c). The background luminescence was obtained from cultures of M. smegmatis with the empty pMV306hsp. Assays were performed with three independent mid-log 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: To achieve the highest signal to background ratio we tested different integration times (0.1–10 s) for the three reporters, at four different substrate concentrations for Gluc and FFluc (Fig. 5). As shown in Fig. 5A, increasing the integration time yielded an increased ratio for FFluc, reaching a maximal ratio at integration times between 5 and 10 s, depending on substrate concentration. This was also the case for Lux, with 10 s yielding the best result (Fig. 5C). In contrast, increasing the integration time resulted in a decreased signal to noise ratio for Gluc (Fig. 5B). This was likely due to the high background produced by auto-oxidation of its substrate coelenterazine [47]. Therefore, for further studies the integration times selected were 5 s, 0.1 s and 10 s for FFluc, Gluc and Lux respectively.


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)

Signal:noise for FFluc and Lux increases with integration time, and decreases for Gluc.Luminescence was measured using six different integration times and four different substrate concentrations for M. smegmatis producing FFluc (a) and Gluc (b), or without substrate for Lux (c). The background luminescence was obtained from cultures of M. smegmatis with the empty pMV306hsp. Assays were performed with three independent mid-log 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-g005: Signal:noise for FFluc and Lux increases with integration time, and decreases for Gluc.Luminescence was measured using six different integration times and four different substrate concentrations for M. smegmatis producing FFluc (a) and Gluc (b), or without substrate for Lux (c). The background luminescence was obtained from cultures of M. smegmatis with the empty pMV306hsp. Assays were performed with three independent mid-log 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: To achieve the highest signal to background ratio we tested different integration times (0.1–10 s) for the three reporters, at four different substrate concentrations for Gluc and FFluc (Fig. 5). As shown in Fig. 5A, increasing the integration time yielded an increased ratio for FFluc, reaching a maximal ratio at integration times between 5 and 10 s, depending on substrate concentration. This was also the case for Lux, with 10 s yielding the best result (Fig. 5C). In contrast, increasing the integration time resulted in a decreased signal to noise ratio for Gluc (Fig. 5B). This was likely due to the high background produced by auto-oxidation of its substrate coelenterazine [47]. Therefore, for further studies the integration times selected were 5 s, 0.1 s and 10 s for FFluc, Gluc and Lux respectively.

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