Optimisation of bioluminescent reporters for use with mycobacteria

• Published in: PloS one Vol. 5; no. 5; p. e10777
• Main Authors: Andreu, Nuria, Zelmer, Andrea, Fletcher, Taryn, Elkington, Paul T, Ward, Theresa H, Ripoll, Jorge, Parish, Tanya, Bancroft, Gregory J, Schaible, Ulrich, Robertson, Brian D, Wiles, Siouxsie
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.05.2010; Public Library of Science (PLoS)
• Subjects: Animals; Bacteria; Bacterial infections; Bioluminescence; Biosensors; Chemical reactions; Chromosomes; Codon - genetics; Codons; Drug resistance; Drug screening; E coli; Enzymes; Escherichia coli; Fireflies; Gaussia; Gene Expression; Genes, Reporter - genetics; Genetic Vectors - genetics; Health aspects; Health risks; House mouse; Hygiene; Imaging; Imaging, Three-Dimensional - methods; Immunology; In vivo methods and tests; Infectious diseases; Infectious Diseases/Bacterial Infections; Infectious Diseases/Respiratory Infections; Kinetics; Localization; Luciferase; Luciferases - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Lungs; Medicine; Mice; Microbiology; Microbiology/Medical Microbiology; Molecular Biology; Mycobacterium smegmatis; Mycobacterium smegmatis - cytology; Mycobacterium smegmatis - growth & development; Mycobacterium smegmatis - metabolism; Mycobacterium tuberculosis; Pathogenesis; Pharmaceutical sciences; Plasmids; Public health; Radiology and Medical Imaging; Secretion; Streptococcus infections; Streptococcus mutans; Streptococcus pneumoniae; Technology; Technology application; Tropical diseases; Tuberculosis; Vibrio cholerae; Whole Body Imaging; United Kingdom > UK
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0010777

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. 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. 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.