@article{mbs:/content/journal/jmm/10.1099/jmm.0.001080, author = "Rastogi, Ruchi and Kaur, Gurkamaljit and Maan, Pratibha and Bhatnagar, Archana and Narang, Tarun and Dogra, Sunil and Kaur, Jagdeep", title = "Molecular characterization and immunogenic function of ML1899 (LipG) of Mycobacterium leprae", journal= "Journal of Medical Microbiology", year = "2019", volume = "68", number = "11", pages = "1629-1640", doi = "https://doi.org/10.1099/jmm.0.001080", url = "https://www.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.001080", publisher = "Microbiology Society", issn = "1473-5644", type = "Journal Article", keywords = "Phospholipase", keywords = "Mycobacterium leprae", keywords = "Phagosome", keywords = "Pro-Inflammatory Cytokines", keywords = "Esterase", keywords = "Macrophages", abstract = " 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/β 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.", }