Functional characterization of rv1900c and rv2037c from mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis is one of the most extensively studied human pathogen. The genome of bacterium encode for several hypothetical proteins that needed to be characterized. rv2037c and rv1900c gene is one of them. Rv1900c of M. tuberculosis annotated as LipJ. It is predicted to have two domains, Rv1900NT lipolytic and C-terminal adenylate cyclase domain. Both the genes were expressed under acidic and nutritive stress, in M.tuberculosis H37Ra. In an attempt to characterize Rv2037c, Rv1900c and Rv1900NT functionally, the genes were cloned, expressed and purified from E. coli. The protein demonstrated lipolytic activity with pNP- decanoate as preferred substrate with optimum temperature 40 ºC. In addition, the Rv2037c protein demonstrated phospholipase activity. To understand the effect of genes on mycobacterium physiology, the genes were cloned and expressed in M. smegmatis, a surrogate host. The Rv2037c protein was found in membrane and extracellular fraction, whereas Rv1900c and Rv1900NT were localized in cytoplasmic and extracellular fraction, by western blot analysis. The expression of rv2037c, rv1900c and rv1900NT in M. smegmatis altered colony morphology and cell surface features such as enhanced biofilm and pellicle formation in comparison to control(MS_vec).The expression of genes decreased cell wall permeability, enhanced the TDM content, and resistance against various stresses and antibiotics. MS_Rv2037c,MS_Rv1900c and MS_Rv1900NT demonstrated better infection and intracellular survival capability in infected THP-I macrophage cell. Mice infected with MS_Rv2037c had higher bacterial load in lung, liver, and spleen of infected mice. Rv2037c protein also generated humoral response in EPTB and MDR-TB patients. The counterpart of Rv1900c in M. smegmatis(MSMEG_4477) was knockdown by homologous recombination. Gene knockout(KO) significantly altered colony morphology and growth kinetics of M. smegmatis.The results indicated strongly towards the crucial role of these enzymes in cell wall modulation,infection