| Tuberculosis remains a grave global health threat. About 1/3 of the global population were infected by TB pathogen, Mycobacterium tuberculosis. With the progress of scientific technologies and the development of medical treatments, the biological characteristics of M. tuberculosis have been researched widely and found some treatment medicine. This has made great achievements in curbing the spread of TB. However, the abuse of drugs, HIV co-infection, global transportation and the emergy of drug-resistant bacteria (multidrugs-resistant strains (MDR-TB) and extensively drug-resistant MTB (XDR-TB)) exacerbated the TB predicament. It is urgent to get new antituberculosis drug targets and new anti-TB drugs.Iron is an essential nutrient for almost all biological events. It is vital co-factor for many enzymes, such as various oxygenases, hydroxylases and oxygen-transferring enzymes. It is also involved in many cytochromes that participates in the oxidative phosphorylation and energy production in cells. Deficiency of iron would cause biofunction disorders. So is it in pathogens. However, the iron in host cellular is very limited. The pathogens, such as the Mycobacterium tuberculosis located in macrophages, have to aquire enough iron successfully to development and proliferation.Siderophores, the magic vehicle for pathogens to snatch iron, are small chelators that have high affinity to iron. It's proved that siderophores relate closely to the survival and pathogenicity of pathogens. For instance, the growth of Pseudomonas aeruginosa that couldn't produce siderophore was destroyed seriously in iron-deficiency milieu and it could not cause diseases when infected the burned mice. The development of siderophore-deletion M. tuberculosis was inhibited severely in macrophage. Additionally, Siderophores also involved in the microbial response to oxidative stress. Due to the important roles in M. tuberculosis, siderophores have been the hot model of investigating anti-tuberculosis candidates. Impressively, some effective components have been found.Siderophores in mycobacteria extracellular hydrophilic carboxymycobactin and the cell membrane-related lipophilic mycobactin. The former is secreted to snatch host iron and then transformed into ferric siderophores, ferrin-siderophores are transported into cells by the mycobactins located in the cell envelopes, and then iron ions are delivered into cells to satisfy the physiological events. The biosynthesis of mycobacterium siderophores needs the cooperation of about fourteen enzymes. Though the overall scheme of the siderophore biosynthesis has been established, whether the genes, Rv1344, fadD33and fadE14, involved in the mycobactin synthesis be still controversial.Krithika et al. thought that Rv1345(fadD33) attended in the biosynthesis of mycobactin tail chains and was responsible for activating the lipidic motifs needed. It's reported that FadD33 could promote the development and virulence of MTB in liver and hepatic cells line. Studies of the FadD33 effects on the host siderophore production and other physical functions will expand our understanding of the biosynthesis mechanisms and illuminate potential references to designs of novel anti-tuberculosis drugs.This study mainly focus on the cloning, expression and the functions of fadD33(Rv1345) on the siderophores production, the oxidative stress response and the fatty acids alterations in Escherichia Coli (DE3)and Mycobacterium smegmatis MC2 155. Pairs of primers was designed according to the nucleotide sequence of fadD33(Rv1345) gene in M. tuberculosis H37Rv obtained from the GenBank Database. M. tuberculosis H37Rv genome was used as the template, fadD33(Rv1345) gene was amplified by PCR. The PCR product was ligated to the pMD19-T Simple Vector, and then subcloned into vector pET32a(+) in Escherichia coli and pNIT-myc in Mycobacterium smegmagits. The recombinant plasmids, pET32-Rv1345 and pNIT-myc-Rv1345, were identified through colony PCR and plasmid PCR separately, plasmid restriction enzymes digestion and sequencing. The recombinant plasmid were then transformed separately into E. coli BL21(DE3) and M. smegmatis MC2 155 successfully. The impacts of FadD33 overexpression on the host bacteria, including the growth, siderophore production and oxidative stress tolerance, was explored. And we found that the overexpression of FadD33 promoted the growth of these host and siderophores production in M.smegmatis but had no significant influence in their tolerence to H2O2. Finally, we detected the fatty acids of the recombinant M. smegmatis and the control by GC. The results suggested that FadD33 overexpression alterred the host fatty acids construction.
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