Functional Investigation With The Lipases Of Mycobacterium Tuberculosis

Tuberculosis(TB) is a chronic consumptive of infectious disease caused by M. tuberculosis. An estimated 10 million people developed TB each year in the world, and millions of people died from disease. China is one of the 22 countries with the heaviest burden of TB, ranking as the second after India for the number of TB patients. In recent years, the emergence of multi-drug resistant TB(MDR-TB), extensively drug resistant TB(XDR-TB) and TB/HIV co-infection makes prevention and control of face more serious challenges.The cell envelope of M. tuberculosis is complex and the most striking feature is the extraordinarily high lipid content. M. tuberculosis genome contains 250 genes encoding putative enzymes involved in the synthesis or degradation of lipids. This tends to suggest that lipids and lipid metabolizing enzymes play an important role in the mycobacterial life cycle and also in its virulence. In this study, we focused on M. tuberculosis Lip family members, aiming at digging out the the proteins displaying lipase activity and systematically investigating characteristics of enzymatic activity. We intended to identify the active center sites, and to explore the role of the lipases in mycobacterial pathogenesis. Moreover, we also planned to analyze its immunogenicity, thus providing valuable insight for development of novel vaccines and drugs to control TB.We cloned 14 genes belonging to the lip family of M. tuberculosis H37 Rv and expressed and purified proteins in E. coli. However, all of the products were expressed in insoluble form under different inducing conditions. The lipase activity could not be detected with the refolding products. Given that the recombinant proteins had lost their biochemistry activity in the inclusion form, and it is difficult to estimate the refolding products. Therefore, we used M. smegmatis system to express recombinant proteins. The acetamide-inducible vector was constructed for M. smegmatis expression system on the basis of E. coli-mycobacteria shuttle plasmid pMV261. The genes were expressed and induced in M. smegmatis, and products in soluble form were obtained. The total lipase activities were assayed with the 10 recombinant M. smegmatis. The highest hydrolysis activity on the substrate of long-chain lipids was observed with the LipL protein. The results of the substrate specificity showed that LipL could not only hydrolyze short-chain esters but also had rather high activity on long-chain lipids. Therefore, LipL can be classified as lipase which can hydrolyze water-insoluble lipids. The optimal temperature for lipase enzyme activity was found to be 37°C, and the optimal pH was identified as 8.0. Both of these characteristics reflect the adaptation of mycobacteria to its living environment, i.e., inside the host.The motifs related to the lipase activity were predicted to G359-X-S-X-G363, S88-X-X-K91 or G49-G-G51 based on the bioinformatics analysis. A site-directed mutagenesis experiment was performed, and detection for the lipase activity of the mutants showed that the G50 A, S88 A, and K91 A substitutions resulted in an almost 90% loss of lipase activity compared with the wild-type protein respectively. This result indicated that the lipase activity is related to these two motifs, i.e., the S88-X-X-K91 and G49-G-G51 motifs. Furthermore, a 3D model was built through homologous modeling to obtain insights into the possible location of the active motifs and the catalytic residues among the overall topological organization of Lip L. The 3D model showed that LipL consisted of several α helices and β sheets, and this result was in agreement with the α/β hydrolase-fold family. Meanwhile, a substrate binding pocket was observed at the surface of the G49-G-G51 motif. Structure determines function. These features of the G49-G-G51 motif are consistent with G49-G-G51 motif is the active center and G50 is the key amino acid residues of the active center of the lipase. The overall topological structure of Lip L analyzed by homologous modeling further validated the site-directed mutagenesis experiment results about the active center of the lipase.It was well known that the surface-exposed proteins of bacteria not only play an important role in the pathogenicity, but also are the significant immunogen. The subcellular fractionation of M. smegmatis was separated by differential centrifugation, and the result of the Western blot analysis of the mycobacterial subcellular fractions showed that LipL is located on the cell wall and cell membrane. The localization of this protein suggests that it may play a role in the pathogenicity of mycobacteria, may also be an important immunogen. The LipL protein reacting with the positive sera from TB patients indicated that there were antibodies against LipL produced by the immune system of TB patients. It was suggested that LipL could induce strong humoral response and was expressed by M. tuberculosis during the infecting period in TB patients. The BALB/c mice were immunized with purified product of Lip L protein, the result showed that LipL could induce a strong humoral immune response and activate a CD8+ cell-mediated T cell response. These results demonstrate that Lip L protein is an important immunogen of mycobacteria. Moreover, the mice were challenged intranasally with the overexpression of Lip L in M. smegmatis, and the results showed that colonization ability of the Ms/LipL in mice’s lungs was considerably enhanced compared with the vector control strain. Thus this result suggested that the Lip L protein is associated with the pathogenicity of M. tuberculosis.Overall, 14 genes belonging to the Lip family of M. tuberculosis were cloned and expressed in this study. The acetamide-inducible vector was constructed for M. smegmatis expression system and a lipase with high enzymatic activity to hydrolyze long-chain lipids was expressed and identified by this expression system. Then, the characterization of LipL lipase activity was analyzed in detail and the active center motif G49-G-G51 of LipL were determined through site-directed mutagenesis and homologous modeling experiments. Finally, we confirmed that LipL is located on the cell wall and cell membrane, and demonstrated that it functions as an immunogen that activates both humoral and cell-mediated responses and might play a role in the pathogenicity of M. tuberculosis.