ABSTRACT
Mycobacterium tuberculosis is known to slow down its transcriptional activity during dormancy. Hence, while using reporter strains, it is important to couple the reporter gene to a promoter that is strong and sensitive both in active and dormant M. tuberculosis. Since respiration is an indispensable process even in dormant bacteria, validation of the promoters of respiratory chain genes - type II NADH dehydrogenase [Pndh] and adenosine triphosphate [ATP] synthase operon [Patps] - of MTB was undertaken for this purpose. Putative promoter containing sequences were cloned upstream of a red fluorescent protein [RFP] gene. Mycobacterium smegmatis or M. tuberculosis carrying episomal constructs were validated for growth, fitness and fluorescence in different models in vitro and in vivo. Either promoter can drive stable and strong expression of RFP in actively growing and dormant M. smegmatis in vitro without significantly affecting growth or viability. Fluorescence due to Pndh and Patps was significantly higher than Phsp60. The fitness of M. tuberculosis H37Rv counterparts was unaffected inside J774 macrophages. In immunocompetent mice, despite an initial attenuation in the lungs, both strains reached loads similar to wild type during chronic infection. In the spleen, the fluorescent strain counts were similar to wild type counts throughout. RFP fluorescence in tissue homogenates was more homogenous among mice due to Pndh compared with Patps. Coupling an appropriate reporter to the promoter of ndh-2 gene of M. tuberculosis can make the reporter expression respiration sensitive and thereby reliably detect both active and dormant populations of the reporter strain.