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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

BLI of gluc-expressing M. smegmatis.Mice were endotracheally inoculated with 3.32×106 CFU of M. smegmatis pMV306hsp+Gluc [two representative mice (M1 and M2) out of three are shown,) or with 1.58×107 CFU of M. smegmatis pMV306hsp as a control (one out of two mice is shown). 10 µg of coelenterazine intranasal was administered 24 h post-inoculation and mice were imaged at time points 0, 5, 10, 15, 30, 60, 120 and 180 min. (a) Images were obtained using an IVIS Spectrum and are displayed as pseudocolour images of peak bioluminescence (given as photons s−1 cm−2 sr−1), with variations in colour representing light intensity at a given location. Integration time was 5 min. (b) Bioluminescence (given as photons s−1) was quantified using the Living image software. (C) 10 µg of coelenterazine was given intraperitoneally to the same mice 5 h post-intranasal coelenterazine. Mice were imaged 0, 5, 10, 15, 20, 25 and 30 min post-intraperitoneal coelenterazine with integration times of 3 min.
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pone-0010777-g012: BLI of gluc-expressing M. smegmatis.Mice were endotracheally inoculated with 3.32×106 CFU of M. smegmatis pMV306hsp+Gluc [two representative mice (M1 and M2) out of three are shown,) or with 1.58×107 CFU of M. smegmatis pMV306hsp as a control (one out of two mice is shown). 10 µg of coelenterazine intranasal was administered 24 h post-inoculation and mice were imaged at time points 0, 5, 10, 15, 30, 60, 120 and 180 min. (a) Images were obtained using an IVIS Spectrum and are displayed as pseudocolour images of peak bioluminescence (given as photons s−1 cm−2 sr−1), with variations in colour representing light intensity at a given location. Integration time was 5 min. (b) Bioluminescence (given as photons s−1) was quantified using the Living image software. (C) 10 µg of coelenterazine was given intraperitoneally to the same mice 5 h post-intranasal coelenterazine. Mice were imaged 0, 5, 10, 15, 20, 25 and 30 min post-intraperitoneal coelenterazine with integration times of 3 min.

Mentions: A similar assay was performed using Gluc. Two coelenterazine concentrations were administered intranasally (10 and 20 µg) and images were acquired at different time points over 3 h. A high background was detected in all cases and no differences were observed between mice inoculated with M. smegmatis pMV306hsp+Gluc or M. smegmatis pMV306hsp (Fig. 12A, B). The same mice were then administered 10 µg coelenterazine by the intraperitoneal route 5 h after initial intranasal substrate administration. A high background signal was detected in the abdomen of both the positive and control mice (Fig. 12C), while the bioluminescence observed in the nose was due to the previous intranasal coelenterazine. Consequently, Gluc is not useful for in vivo imaging of M. smegmatis.


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)

BLI of gluc-expressing M. smegmatis.Mice were endotracheally inoculated with 3.32×106 CFU of M. smegmatis pMV306hsp+Gluc [two representative mice (M1 and M2) out of three are shown,) or with 1.58×107 CFU of M. smegmatis pMV306hsp as a control (one out of two mice is shown). 10 µg of coelenterazine intranasal was administered 24 h post-inoculation and mice were imaged at time points 0, 5, 10, 15, 30, 60, 120 and 180 min. (a) Images were obtained using an IVIS Spectrum and are displayed as pseudocolour images of peak bioluminescence (given as photons s−1 cm−2 sr−1), with variations in colour representing light intensity at a given location. Integration time was 5 min. (b) Bioluminescence (given as photons s−1) was quantified using the Living image software. (C) 10 µg of coelenterazine was given intraperitoneally to the same mice 5 h post-intranasal coelenterazine. Mice were imaged 0, 5, 10, 15, 20, 25 and 30 min post-intraperitoneal coelenterazine with integration times of 3 min.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010777-g012: BLI of gluc-expressing M. smegmatis.Mice were endotracheally inoculated with 3.32×106 CFU of M. smegmatis pMV306hsp+Gluc [two representative mice (M1 and M2) out of three are shown,) or with 1.58×107 CFU of M. smegmatis pMV306hsp as a control (one out of two mice is shown). 10 µg of coelenterazine intranasal was administered 24 h post-inoculation and mice were imaged at time points 0, 5, 10, 15, 30, 60, 120 and 180 min. (a) Images were obtained using an IVIS Spectrum and are displayed as pseudocolour images of peak bioluminescence (given as photons s−1 cm−2 sr−1), with variations in colour representing light intensity at a given location. Integration time was 5 min. (b) Bioluminescence (given as photons s−1) was quantified using the Living image software. (C) 10 µg of coelenterazine was given intraperitoneally to the same mice 5 h post-intranasal coelenterazine. Mice were imaged 0, 5, 10, 15, 20, 25 and 30 min post-intraperitoneal coelenterazine with integration times of 3 min.
Mentions: A similar assay was performed using Gluc. Two coelenterazine concentrations were administered intranasally (10 and 20 µg) and images were acquired at different time points over 3 h. A high background was detected in all cases and no differences were observed between mice inoculated with M. smegmatis pMV306hsp+Gluc or M. smegmatis pMV306hsp (Fig. 12A, B). The same mice were then administered 10 µg coelenterazine by the intraperitoneal route 5 h after initial intranasal substrate administration. A high background signal was detected in the abdomen of both the positive and control mice (Fig. 12C), while the bioluminescence observed in the nose was due to the previous intranasal coelenterazine. Consequently, Gluc is not useful for in vivo imaging of M. smegmatis.

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