Bandana Kumari
The genome of Mycobacterium tuberculosis encoded for several hypothetical proteins that needed to be characterized. rv2037c gene was marked as hypothetical and was conserved in only pathogenic strains of mycobacteria, while it was absent in non-pathogenic M. smegmatis. The gene expression was 25 and 4 folds upregulated under acidic and nutritive stress, respectively in M. tuberculosis H37Ra. In an attempt to characterize Rv2037c functionally, the gene was cloned, expressed and purified from E. coli. The protein demonstrated lipolytic activity with pNP-decanoate as preferred substrate with optimum pH 8.0 and temperature 40 ºC. In addition, the protein demonstrated phospholipase activity. Predicted active sites for protein were confirmed by site directed mutagenesis followed by enzyme assay for activity loss. To understand the effect of this gene on mycobacterium physiology, the gene was cloned and expressed in M. smegmatis, a surrogate host. The expression of rv2037c in M. smegmatis (MS_Rv2037c) altered colony morphology and cell surface features such as enhanced biofilm and pellicle formation in comparison to control (MS_vec). MS_Rv2037c decreased cell wall permeability, enhanced the TDM content, and resistance against various stresses and antibiotics. MS_Rv2037c demonstrated better infection and intracellular survival capability in infected THP-I macrophage cell. Rv2037c induced the production of pro-inflammatory cytokines TNF-α and IL-12 in infected
mice, suggesting its role in immune modulation. Recombinant protein also generated humoral response in EPTB and MDR-TB patients. The results indicated strongly towards the crucial role of this enzyme in cell wall modulation, infection and intracellular survival of mycobacterium