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In vitro  mimicking conditions are thought to reflect the environment experienced by  Mycobacterium tuberculosis  inside the host granuloma. The majority of  in vitro  dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of  M. tuberculosis  from south India in addition to H37Rv for our  in vitro  oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for  in vitro  stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4 +  central memory T cells in the circulation of HHC compared to PTB ( p  < 0.005 for ESAT-6 and  p  < 0.0005 for Lpd). This shows proteins that are predicted to be up regulated during  in vitro  hypoxia in most prevalent clinical strains would indicate possible potential immunogens.  In vitro  hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms.

frontiers in microbiology

Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen’s Specific Memory T Cells in Healthy Household Contacts