Mycobacterium tuberculosis was first isolated more than 125 years ago. Although a huge amount of research has been devoted to it over this time, tuberculosis still represents a major public health threat in many countries. The main hindrances in fighting this disease include a lack of understanding of the human infection, its establishment and progression, as well as the host-pathogen interactions that determine the different outcomes. Furthermore, the treatment regimen of six months administration of up to four drugs has not evolved in more than four decades, and recent years have seen an alarming increase in multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains. It is clear then that novel and imaginative approaches are needed to speed up both basic and translational research in tuberculosis, especially in the areas of vaccine and drug development.
Optimisation of Bioluminescent Reporters for Use with Mycobacteria. 2010 PLoS ONE 5(5): e10777. 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.