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.

In vivo imaging of treatment responses at the molecular level could have a significant impact on the speed of drug discovery and ultimately lead to personalized medicine. Strong interest has been shown in developing quantitative fluorescence-based technologies with good molecular specificity and sensitivity for noninvasive 3D imaging through tissues and whole animals. We show herein that tumor response to chemotherapy can be accurately resolved by fluorescence molecular tomography (FMT) with a phosphatidylserine-sensing fluorescent probe based on modified annexins. We observed at least a 10-fold increase of fluorochrome concentration in cyclophosphamide-sensitive tumors and a 7-fold increase of resistant tumors compared with control studies. FMT is an optical imaging technique developed to overcome limitations of commonly used planar illumination methods and demonstrates higher quantification accuracy validated by histology. It is further shown that a 3-fold variation in background absorption heterogeneity may yield 100% errors in planar imaging but only 20% error in FMT, thus confirming tomographic imaging as a preferred tool for quantitative investigations of fluorescent probes in tissues. Tomographic approaches are found essential for small-animal optical imaging and are potentially well suited for clinical drug development and monitoring.

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the P_mtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.