Molecular characterization and immunogenic function of ML1899 (LipG) of Mycobacterium leprae.

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

Cell Wall Associated Factors of Mycobacterium tuberculosis as Major Virulence Determinants: Current Perspectives in Drugs Discovery and Design.

BACKGROUND: Mycobacteria genus is responsible for deadly diseases like tuberculosis and leprosy. Cell wall of bacteria belonging to this genus is unique in many ways. It plays a major role in the pathogenesis and intracellular survival inside the host. In intracellular pathogens, their cell wall acts as molecular shield and interacts with host cell milieu to modulate host defense responses. OBJECTIVES: In this review, we summarize the factors that participate in the biosynthesis of unique mycobacterial cell wall, understand their potential as drug targets and the recent developments where they have been evaluated as possible drug targets. RESULTS: Several cell wall associated factors that play crucial roles in the synthesis of cell wall components like Antigen 85 complex, Glycosyltransferases (GTs), LM (lipomannan) and LAM (lipoarabinomannan), mAGP Complex, lipolytic enzyme have been categorically documented. Most of the presently used anti TB regimens interrupted cell wall synthesis, but the emergence of drug resistant strains made it mandatory to identify new drug targets. Novel drug candidates which could inhibit the synthesis of cell wall components have been thoroughly studied worldwide. CONCLUSION: Studies demonstrated that the cell wall components are unique in terms of their contribution in mycobacterium pathogenesis. Targeting these can hamper the growth of M. tuberculosis. In this study, we scrutinize the drugs under trials and the potential candidates screened through in silico findings.