title: Coupling reporter expression to respiration detects active as well as dormant mycobacteria in vitro and in mouse tissues.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India. Electronic address: subashbt13@gmail.com.
authors.forename: Subash Chand
authors.initials: SC
authors.lastname: Verma
authors.affiliation: Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India. Electronic address: mag.bacillus@gmail.com.
authors.forename: Umamageswaran
authors.initials: U
authors.lastname: Venugopal
authors.affiliation: Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India. Electronic address: sahebshan11@gmail.com.
authors.forename: Shaheb Raj
authors.initials: SR
authors.lastname: Khan
authors.affiliation: Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India; Academy of Scientific and Innovative Research, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India. Electronic address: sohail@cdri.res.in.
authors.forename: Md Sohail
authors.initials: MS
authors.lastname: Akhtar
authors.affiliation: Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India; Academy of Scientific and Innovative Research, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India. Electronic address: manju@cdri.res.in.
authors.forename: Manju Yasoda
authors.initials: MY
authors.lastname: Krishnan
keywords: Dormancy; Mycobacterium tuberculosis; Promoters; Reporter genes; Respiratory chain