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

The in vivo induced antigen technology (IVIAT)(1) has been used for the identification of open reading frames (ORFs) which could be possible therapeutic targets. A recombinant lambdagt11:: Mycobacterium tuberculosis H37Rv expression library was screened with pooled TB patient sera preabsorbed with in vitro grown M. tuberculosis H37Rv. Preabsorption of pooled TB patient sera allowed identification of antigens specifically expressed or upregulated during infection and growth in vivo. Six ORFs were identified, of which four (rv0287, rv2402, rv3878 and rv1045) were of hypothetical functions. Rv0287 is a probable regulatory protein. Rv3878 is present uniquely in M. tuberculosis H37Rv and is a part of RDI deletion region of M. bovis BCG, which includes esat 6 region. This could be exploited as a tool for diagnosis. Two ORFs were assigned function solely on the basis of homology, dnaQ (rv3711c) and lpdA (rv3303c). dnaQ codes for the epsilon subunit of DNA polymerase III, which is responsible for the proofreading activity of the complex. lpdA codes for dihydrolipoamide dehydrogenase, which is a part of many multienzyme complexes such as pyruvate dehydrogenase, keto-acid dehydrogenase and alpha-ketoglutarate dehydrogenase. These two enzymes appear to be potential targets for drug development.

Bioluminescent Mycobacterium aurum expressing firefly luciferase for rapid and high throughput screening of antimycobacterial drugs in vitro and in infected macrophages.

The slow growth and highly infectious nature of Mycobacterium tuberculosis is a limiting factor in its use as test organism in high throughput screening for inhibitory compounds. To overcome these problems, use of surrogate strains and reporter genes have been considered. In this study, we have investigated the application of a fast growing nonpathogenic M. aurum expressing firefly luciferase in rapid screening of antituberculosis compounds in vitro and in infected macrophages using bioluminescence assay. The assay is based on luminescence determination using luciferin as substrate. Inhibition of bioluminescence was obtained with frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their reported MICs. Inhibition could be observed as early as 2 h in vitro and within 24 h in infected macrophages. The system can reliably be used in high throughput screening.

Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages.

The development of new drugs against Mycobacterium tuberculosis is impeded by slow growth and highly infectious nature of the organism that warrants the need to work under highly stringent biosafety conditions. These problems can be overcome by use of reporter genes and surrogate strains. A strain of rapidly growing M. aurum has been recommended as test organism to screen inhibitors of mycobacteria to preselect compounds for progression into testing against M. tuberculosis. We have investigated the application of recombinant M. aurum expressing green fluorescent protein in rapid screening of antituberculosis compounds in vitro and in infected macrophages. Recombinant M. aurum[pGFM-11] expressing green fluorescent protein was constructed. The assay is based on measurement of fluorescent intensity at 509 nm. A good correlation was found between fluorescence and growth. Fluorescence of recombinant M. aurum was inhibited in vitro within 8 to 24 h by frontline antimycobacterial drugs at their reported MICs whereas inhibition in infected macrophages was observed in 72 h. Therefore green fluorescent reporter system provides a convenient screen to test antimycobacterial compounds that are active in vitro and within infected macrophages.