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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Optimisation of bioluminescent reporters for use with mycobacteria.

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes.

The origin, diversity, and structure function relationships of insect luciferases.

Tuberculosis continues to be a major global public health problem. In addition, the increasing prevalence of multidrug-resistant Mycobacterium tuberculosis is of concern (20) and has fostered a sense of urgency with regard to the need to acquire new drugs. Several rapid methods such as the microplate Alamar Blue assay (MABA) (3, 5), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based assay (9), and the luciferase assay (1, 2, 7, 21) have been established for the screening of the antimycobacterial activities of compounds. However, each has one or more drawbacks for high-throughput screening, including the need to add a dye or a substrate to test the viability of the bacteria, a step which both increases labor and decreases safety.

Fluorometric assays based on the Aequorea victoria gfp gene encoding a green fluorescent protein (GFP) or its mutants have been used widely in prokaryotes (8, 10, 11, 24) due to several properties of gfp that are advantageous for use as a reporter for bacterial viability and growth, including the low level of toxicity, continuous production during replication, and easy imaging and quantification (11, 25). Previous studies with mycobacteria exclusively used the hsp60 promoter, which has been fused to gfp in a variety of investigations (8, 11, 26).

Collins et al. (6) demonstrated that a recombinant M. tuberculosis strain carrying pFPV2, an hsp60::gfp construct, could be used to assess the MICs of known antimycobacterial compounds. However, the low level of the fluorescence signal and the relatively high background autofluorescence of the medium precluded the establishment of a robust assay that could be used with confidence for high-throughput screening.

Previous investigators (6, 13, 17, 18, 23) have identified a highly inducible promoter sequence which regulates the expression of the acetamidase gene in M. smegmatis NCTC 8159 and which demonstrated a high level of gene expression in the presence of amide inducers such as acetamide.

In this study we demonstrate that an acetamidase promoter from M. smegmatis strain mc2155 permits constitutive, high-level gfp expression in M. tuberculosis strains H37Ra, H37Rv, and Erdman, resulting in a fluorescence signal significantly higher than that which is achievable with the hsp60 promoter and facilitating the determination of MICs of antimicrobial agents without the addition of reagents postincubation.

Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

Summary


In this study, we have established conditions for generating Leishmania amazonensis recombinants stably expressing the firefly luciferase gene. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing the course of the parasitism to be readily monitored in real time in the living animals such as laboratory mice. First, a model was established, using parasite-infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracel-lular amastigote-harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. These assays prove that the real-time bioluminescent assay is not only sensitive but also more rapid than culture-base techniques allowing to monitor parasite-load before any clinical signs of leishmaniasis are detectable. In short, this luciferase imaging study is useful to monitor the efficacy of anti-leishmanial drugs on live cell culture and to trace leishmanial infection in animal models.

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Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice.

Functional relationships between proteins that do not share global structure similarity can be established by detecting their ligand-binding-site similarity. For a large-scale comparison, it is critical to accurately and efficiently assess the statistical significance of this similarity. Here, we report an efficient statistical model that supports local sequence order independent ligand–binding-site similarity searching. Most existing statistical models only take into account the matching vertices between two sites that are defined by a fixed number of points. In reality, the boundary of the binding site is not known or is dependent on the bound ligand making these approaches limited. To address these shortcomings and to perform binding-site mapping on a genome-wide scale, we developed a sequence-order independent profile–profile alignment (SOIPPA) algorithm that is able to detect local similarity between unknown binding sites a priori. The SOIPPA scoring integrates geometric, evolutionary and physical information into a unified framework. However, this imposes a significant challenge in assessing the statistical significance of the similarity because the conventional probability model that is based on fixed-point matching cannot be applied. Here we find that scores for binding-site matching by SOIPPA follow an extreme value distribution (EVD). Benchmark studies show that the EVD model performs at least two-orders faster and is more accurate than the non-parametric statistical method in the previous SOIPPA version. Efficient statistical analysis makes it possible to apply SOIPPA to genome-based drug discovery. Consequently, we have applied the approach to the structural genome of Mycobacterium tuberculosis to construct a protein–ligand interaction network. The network reveals highly connected proteins, which represent suitable targets for promiscuous drugs.

Contact: [email protected]

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A unified statistical model to support local sequence order independent similarity searching for ligand-binding sites and its application to genome-based drug discovery

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach

Bioluminescent Mycobacterium aurum Expressing Firefly Luciferase for Rapid and High Throughput Screening of Antimycobacterial Drugs in Vitro and in Infected Macrophages

D.K. Deb

Papers

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach

Bioluminescent Mycobacterium aurum Expressing Firefly Luciferase for Rapid and High Throughput Screening of Antimycobacterial Drugs in Vitro and in Infected Macrophages