Analysis Of Wag31 - Mediated Mycobacterium Drug Resistance And Wag31 DNA Vaccine

Tuberculosis, especially drug resistant tuberculosis (DR-TB), is a serious threat to global human health. The report about patients with totally drug resistant tuberculosis (TDR-TB) in India, is a wake-up call to the world that TB had taken a turn for the worse. More and more research on the mechanism of tuberculosis drug resistance is needed urgently, once drug-sensitive tuberculosis turn to drug resistant tuberculosis, patients had to take second-line drugs which is expensive, less effective and more toxic, and a many more months drug treatment. Drug resistance research and novel immunotherapy treatment are the two pathways which expected to solve those problems. We intended to look for clues from extensively drug-resistant tuberculosis (XDR-TB) Proteomics. Using isobaric tag for relative and absolute quantitation (iTRAQ), we found an overexpressing protein wag31 (Rv2145c) from the different expressed proteins between H37Rv and two XDR-TBs. Wag31 was proved to be involved with Cell morphology and peptidoglycan synthesis. We made several M. smegmatis strains with different wag31 expressing levels separately, and we verified the conclusions of previous studies that the overexpression of wag31 caused cell enlarging and cell lysis. In addition, we described knock-down of wag31 expression caused cell lengthening and a lower Asymmetry level of cell division.Then we tested Minimum inhibitory concentration (MIC) of wag31 knock-down strain, it showed that MIC of wag31 knock-down strains was 1/4 of control strains against Rifampicin and Erythromycin,1/2 of control strains against Clofazimine and novobiocin. In addition, Bacterial permeability analysis showed that knock-down of wag31 Improved cell permeability of lipid and decreased cell permeability of Ethidium bromide. It suggested that the Improved the sensitivity might related to Lipid permeability changes.Subsequently, to investigate the function of wag31 in vivo, CoIP was used to identify the wag31 Binding proteins. Several proteins we found are involved in mycolic acid synthesis, which are Rv3285 (AccA3), Rv3799c (AccD4), Rv3280 (AccD5), Rv2524c (fas), Rv1484 (inhA) and two ATPases.Since the function of Rv3285 on long chain lipid synthesis and mycolic acid had been proved, we overexpressed Rv3285 in M. smegmatis strains and we found Rv3285 overexpression decreased the lipid permeability and improved the MIC by 2 times against rifampicin and novobiocin. It demonstrated that wag31 maintains the resistance against lipophilic drugs though controlling the lipid permeability of cell wall and Rv3285 is involved with this mechanism.Besides that, we tested the Immune protection on mouse of two DNA vaccine, pVax-wag31 and pVax-Ag85a+wag31. It shows that only wag31 can increase the ratio of CD4+ T cell and CD8+ T cell in Peripheral blood lymphocytes. Ag85a and wag31 can induce high IFN-gamma production of Spleen lymphocytes. Besides that, both two proteins induced very low level of IL-4 of Spleen lymphocytes. It suggests that DNA vaccine pVax-Ag85a+wag31 can Stimulate Peripheral blood T lymphocyte maturation, enhance IFN-gamma production and Thl Immune response, which indicates that pVax-Ag85a+wag31 is a potential Effective Protective and therapeutic vaccine.