Optimisation of Bioluminescent Reporters for Use with Mycobacteria

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 FindingsOne 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/SignificanceWhile 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.

Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex

“…Additionally, M. bovis BCG lux requires containment level (CL) 2, rather than CL3 laboratories. This organism has been very well-characterized and utilized in a wide range of clinical and non-clinical studies [21,24,27–32,51]. For example, differences in M. bovis BCG lux RLU measurements have been shown to reflect changes in the immune response in human whole blood and cellular models, including differences between tuberculin-positive and -negative individuals [23], changes induced by HIV infection and following antiretroviral treatment [25,26], changes in mycobacterial growth limitation induced by vaccines [24] and Vitamin D supplementation in vitro and in a clinical trial [29–31], and differences in gene expression in immune biological pathways compared to M. tuberculosis [32].…”

“…For example, differences in M. bovis BCG lux RLU measurements have been shown to reflect changes in the immune response in human whole blood and cellular models, including differences between tuberculin-positive and -negative individuals [23], changes induced by HIV infection and following antiretroviral treatment [25,26], changes in mycobacterial growth limitation induced by vaccines [24] and Vitamin D supplementation in vitro and in a clinical trial [29–31], and differences in gene expression in immune biological pathways compared to M. tuberculosis [32]. In addition we have also evaluated luminescent mycobacteria in the mouse model [27]. Hence the use of M. bovis BCG lux in in vivo studies as a model organism for M. tuberculosis lends further benefit to in vivo assays using G. mellonella .…”

Bacterial vectors for imaging and cancer gene therapy: a review

“…For example, strains of E. coli rendered metabolically dependent on diaminopimelic acid (DPI) have been generated, permitting maintenance of plasmids featuring DPI-producing genes as plasmid loss lead to bacterial cell death. 68,69 Transposon, 46,70 transducing bacteriophages, 42 temperature sensitive vectors 71 and bacteriophage integrase systems [72][73][74] have been developed to integrate genes into the chromosomes of selected bacteria. However, it is important to stress that the maintenance and expression of high levels of recombinant DNA may place an unwelcome metabolic burden microorganisms.…”

“…78 Furthermore, codon optimizing the firefly luciferase for M. tuberculosis resulted in a 30-fold increase in signal. 74 However codon optimization may have unforeseen effects; the lux operon codon optimized for M. tuberculosis was found to be nonfunctional 74 reported to be as a result of secondary DNA structures which impede translation.…”

“…For bacteria expressing the lux operon, it is possible that the availability of aldehyde and FMNH 2 are limiting factors. The insertion of an extra promoter in front of luxCDE to boost substrate synthesis resulted in a six-fold higher lux signal in M. smegmatis 74 and S. aureus. 79 In yeast, co-expression of luxAB together with an frp gene which encodes an NADPH-FMN oxidoreductase from V. harveyi, led to a 100-fold increase in luminescence.…”