A novel differential expression system for gene modulation in Mycobacteria.
Tuberculosis (TB) remains a major global health problem, and successful genetic manipulation of mycobacteria is crucial for developing new approaches to study the mechanism of pathogenesis of Mycobacterium tuberculosis (M.tb) and to combat TB. In this study, a series of M.tb furA gene operator/promoter (pfurA) mutants were generated aiming at optimization of the promoter activities in mycobacterial strains. Measured by the lacZ gene-fusion reporter system, change of the initial codon GTG to the preferred ATG resulted in a double increase of beta-galactosidase activity, while a 6-bp substitution in the conserved FurA binding AT-rich region upstream of furA gene led to 4-6 folds increase of the activity. It is significant that combination of both mutations showed about 10 folds of beta-galactosidase activity higher than that of the prototype pfurA. Furthermore, all of the furA promoters were expressed continuously in vivo during intracellular growth of Mycobacterium bovis BCG, and were induced early upon infection in macrophages. Employing the series of pfurA-based differential expression vectors, M.tb chimeric antigen Ag856A2 known for its excellent immunogenicity, was shown to be expressed at different levels in the recombinant Mycobacterium smegmatis and BCG strains. These results indicated that this differential expression system is feasible to express any target antigen of interest in a modular fashion for the study of gene regulation in mycobacterial strains, and also for the development of different recombinant BCG vaccine candidates against TB or other infectious diseases, which would be beneficial for elicitation of optimal immune response.
Fan
Xiao-Yong
XY
Department of Microbiology, School of Life Sciences, Fudan University, Shanghai, China. xyfan008@hotmail.com
Ma
Hui
H
Guo
Jian
J
Li
Zhong-Ming
ZM
Cheng
Zhi-Heng
ZH
Guo
Sheng-Qi
SQ
Zhao
Guo-Ping
GP
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics* / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D001616:beta-Galactosidase