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

In vitro mimicking conditions are thought to reflect the environment experienced by M. tuberculosis inside the host granuloma. The majority of the in vitro dormancy experimental models used laboratory adapted strain H37Rv or Erdman strain over the 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 the hypoxic cultures, resolved by two-dimensional electrophoresis (2-DE) and protein spots were characterized by mass spectrometry. Totally 49 spots were characterized as over-expressed or newly appeared between the 3 strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these 2 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). Multicolour flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in circulation of HHC when compared to PTB (p<0.005 for ESAT-6 and p<0.0005 for Lpd). This shows proteins that are predicted to be upregulated during in vitro hypoxia in most prevalent clinical strains would bring the possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also bring the probable true representative antigens that involved during adaption mechanism

Biophysical and biochemical characterization of Rv3405c, a tetracycline repressor protein from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, is one among the deadliest pathogens in the world. Due to long treatment regimen, HIV co-infection, persistence of bacilli in latent form and development of XDR and TDR strains of Mtb, tuberculosis has posed serious concerns for managing the disease, and calls for discovery of new drugs and drug targets. Using a computational pipeline involving analysis of the structural models of the Mtb proteome and an analysis of the ATPome, followed by a series of filters to identify druggable proteins, solubility and length of the protein, several candidate proteins were shortlisted. From this, Rv3405c, a tetR family of DNA binding protein involved in antibiotic resistance, was identified as one of the good drug targets. Rv3405c binds to the upstream non coding region of Rv3406 and causes repression of Rv3406 activity there by affecting the downstream processes involved in antibiotic resistance was further characterized. The Rv3405c gene was cloned; the gene product was over-expressed in E. coli and purified by Ni NTA chromatography. DNA binding studies by EMSA showed that the recombinant Rv3405c protein binds to the DNA sequence corresponding to the promoter region of Rv3406 and upon addition of tetracycline, the DNA binding activity was lost. P-galactosidase reporter assay in E. coli using both wild type and a DNA binding defective mutant protein indeed proved that Rv3405c acts as a repressor. (C) 2018 Elsevier Inc. All rights reserved