Function And Transcriptional Regulation Of M.Tuberculosis Transcriptional Regulator Whib2

The incidence of Tuberculosis, a serious threat to human health, keeps increasing. 2010, it estimated that there was 8.8 million new cases of TB patients,1.1 million patients deaths of HIV-infected TB, and another 350,000 died of AIDS co-infection with tuberculosis. With the development of human society and drug treatment, the biological characteristics of the TB pathogen-Mycobacterium tuberculosis has been further research, then developed therapeutic drugs, such as streptomycin, isoniazid, rifampin and so on. This has made great achievements of TB control, limited the spread of TB. From 1995 to 2010, with the implementation of modern TB control strategy/ stop TB strategy,55 million cases of TB patients have been treated,46 million cases of patients have been treated successfully, the lives of nearly 700 million have been saved. However, due to the unreasonable application of chemical drugs, AIDS co-infection with tuberculosis, the increase of population mobility and the prevalence of drug-resistant TB bacteria, the decline speed of TB epidemic in recent years have been changed more slowly, even rebounded in some areas. New tuberculosis drug targets and anti-tuberculosis medicines are urgently needed to combat the rampant tuberculosis.whi genes exist largely in Actinomyces and Mycobacterium, named after the mutations turning Streptomyces coelicolor colonies into white. whiB genes belong to a subclass of whi, involving in a wide range of events, such as cell division, spore formation, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. WBL are predicted to be transcription factor based on the helix-turn-helix （HTH） motif by Protein Sequence Analysis （PSA） server. M. tuberculosis WhiB family contains seven members, namely WhiB1～7. WhiB protein has four conserved cysteine residues arranged as the C-X19-36-CXXC-X5-7-C. The CXXC can coordinate [2Fe-2S]. Except WhiB5 and WhiB6, other five WhiB proteins of M. tuberculosis are highly conserved among the Mycobacterium based on comparative genomic analysis. But, the function of the 7 WBL in M. tuberculosis have not annotated. Incubated with insulin to detect protein disulfide reduction function, the results showed that except WhiB2, the other six WhiB proteins were able to restore the disulfide bonds of insulin. WhiB1 is the only one that regulated by the cAMP receptor protein CRP protein in M. tuberculosis. It can interact with glgB. WhiB3 is associated with tissue injury. M. tuberculosis regulate the expression of whiB3 to response to environmental signals in vivo. M. tuberculosis H37Rv whiB7 null mutant strains were highly sensitive to many antibiotics （such as tetracyclines-tetracycline, aminoglycosides-streptomycin, macrolides-erythromycin）. It suggested that whiB7 is relative to pressure sensing. Isoniazid, ethambutol and cycloserine can induce whiB2 transcription. Aminoglycosides （streptomycin and kanamycin） can upregulate whiB7. Exposure to acidic condition （pH4.5） can upregulate M. tuberculosis whiB6 and whiB3.WhiBTM4 （the whiB homolog in TM4 mycobacteriophage） overexpressed M. smegmatis showed a WhiB2 knockout phenotype, namely the blockage of membrane formation, indicating that WhiBTM4 can downregulate WhiB2. M. tuberculosis whiB2 is a homolog of whmD. M. smegmatis whmD is an essential gene. Deletion of chromosomal whmD led to irreversible, branched growth with diminished septum formation, suggestive of a role in cell division. It suggested that WhiB2 was relative to cell division, but the mechanism have no evidence.In order to research the function of WhiB2, the M. tuberculosis H37Rv whiB2 was cloned into shutter plasmid pNIT-myc so that the recombinant plasmid pNIT-myc-whiB2 was been gotten. Then, the recombinant plasmid was transferred into Mycobacterium smegmatis MC2155. Western-blot verified the whiB2 expressed successfully. Then, the impact of overexpression of whiB2 in M. smegmatis MC2155, comprise of the growth, anti-oxidative stress and expression of the gene that is relative to cell division was been researched. Also, we studied the MIC of the antimycobacteria to recombinant M. smegmatis MC2155. The result shows that overexpresssion of whiB2 has no effect on growth, but more sensitive to isoniazide and capromycin. However, the recombinant M. smegmatis MC 155 show more tolerance to peroxide. The result of RT-PCR show that WhiB2 upregulate MSMEG₄225 and ftsZ. In short, our research suggested the relationship between overexpression of whiB2 and antibiotic and anti-oxidative stress. Moreover, our results revealed the mechanism why whiB2 is relative to cell division. It is good for understand the mechanism of physiology and biochemistry in M. tuberculosis...