Mycobacterium Tuberculosis Lipid Arab Mannan Biosynthetic Pathway Gene Cloning And Functional Studies

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most effective bacterial human pathogen. Over one-third of the world's population is infected with M. tuberculosis and almost 2 million people die from tuberculosis annually. Compounding the problem, strains of M. tuberculosis that are resistant to the major drugs used to treat tuberculosis are rapidly emerging worldwide. The eradication of tuberculosis requires the development of novel anti-mycobacterial agents for therapeutic treatment of M. tuberculosis, the etiological agent of tuberculosis. The mycobacterial cell wall is a complex mixture of unique components that sets mycobacteria apart from other typical bacterial species and is the site of action of many of the first-line antimycobacterial agents. Thus the biosynthetic pathways leading to formation of key mycobacterial cell wall are attractive targets for the rational design of new antituberculosis agents.Phosphatidyl-myo-inositol and metabolically derived products such as phosphatidyl-myo-inositol-mannosides, lipomannan, and lipoarabinomannans are prominent and important phospholipids/lipoglycans in the cell wall of mycobacterial species. Phosphatidyl-myo-inositol andphosphatidyl-myo-inositol-mannosides are believed to play essential roles in the membrane stability and thus for cell viability. Lipoarabinomannans, especially mannose-capped LAM, has been implicated in various immunomodulatory activities including inhibiting of the activation of the macrophages, inhibiting of the production of IL-12 by macrophages and dendritic cells, and modulating of M. tuberculosis-induced macrophage apoptosis. All these activities are likely to favor the intra-macrophagic survival of the bacilli.The pimA gene of mycobacteria encodes a a-mannosyltransferase involved in the transfer reaction of the very first mannose from GDP-Mannose to the carrier lipid phosphatidyl-myo-inositol, a precursor in the synthesis of lipoarabinomannans. pimA has been proposed to play an essential role in growth of mycobacteria. In this study,the pimA gene from M. tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the PimA enzyme in fusion with a histidine-rich peptide on the N-terminal. The PimA-His tag fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purity and molecular weight were determined by high performation liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). Its correct folding was verified by circular dichrosim spectroscopy (CD) study. The enzyme assays revealed that PimA showed a requirement for Mg²⁺ for the activity, and the K_m and V_max values of PimA enzyme were estimated at 18μM and 0.1 nmol/min/μg, respectively.Inositol monophosphatase is an enzyme in the biosynthesis of myo-inostiol, a crucial substrate for the synthesis of phosphatidylinositol, which has been demonstrated to be an essential component of mycobacteria. In this study, the Rv2131c gene from M. tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into E. coli BL21 (DE3) strain, allowing the expression of the enzyme in fusion with a histidine-rich peptide on the N-terminal. The fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purified Rv2131c gene product showed inositol monophosphatase activity but with substrate specificity that was broader than those of several bacterial and eukaryotic inositol monophosphatases, and it also acted as fructose-1, 6-bisphosphatase. The dimeric enzyme exhibited dual activities of inositol monophosphatase and fructose-1, 6-bisphosphatase, with K_m of 0.22 ± 0.03 mM for inositol-1-phosphate and K_m of 0.45 ± 0.05 mM for fructose-1,6-bisphosphatase. To better understand the relationship between the function and structure of the Rv2131c enzyme, we constructed D40N, L71A, and D94N mutants and purified these corresponding proteins. Mutations of D40N and D94N caused the proteins to almost completely lose both the inositol monophosphatase and fructose-1, 6-bisphosphatase activities. However, L71A mutant did not cause loss either of the activities, but the activity toward the inositol was12-fold more resistant to inhibition by lithium (IC₅₀ = 60 mM). Based on the substratespecificity and presence of conserved sequence motifs of the M. tuberculosis Rv2131c, we proposed that the enzyme belonged to class IV fructose-1, 6-bisphosphatase (FBPase IV).