Mechanisms Of Virulence Attenuation In Mycobacterium Tuberculosis H37Ra

Mycobacterium tuberculosis (Mtb). the causative agent of tuberculosis (TB), is an extraordinarily human pathogen that has latently infected one-third of the world population and causes 9 million new cases and about 1.5～2 million deaths each year world widely. Despite decades of research on chemotherapy for the disease and development of preventive vaccines, it remains a major public-health problem and was declared a global health emergency by WHO in 1993. The emergence of multidrug-resistant Mtb, combining with the HIV/TB co-infection cases, has caused it more urgent to develop new drugs. Therefore, identification of novel virulence factors and revealing of its related molecular mechanism are important scientific endeavous for developing new drug target or therapeutic regiments.Mtb H37Ra is an avirulent relative of the classical laboratory virulent strain H37Rv, both of which are derived from the parent clinical strain H37. To elucidate the mechanisms of virulence attenuation of H37Ra, we compared the genomic sequences between H37Rv and H37Ra, and identified multiple H37Ra-specific variations. The presence of an A219E spontaneous mutation identified in the avirulent Mtb H37Ra led us to characterize the MazG proteins from Mtb H37Rv (MtMazG) and M smegmatis (MsMazG). The biochemical properties of MtMazG were characterized and the A219E substitution was proved to be a loss-of-function mutation with respects to its NTP-PPase activity and the molecular mechanism. The oxidative stress responsive role of MazG and its related mechanism was identified in the mazG null mutant of M. smegmatis. Our research suggests that the A218E mutation in MtMazG might be relevant to the virulence attenuation character of H37Ra.This study includes four parts.In the first part, by using the shotgun sequencing strategy, we obtained the complete genome sequence of M. tuberculosis H37Ra (ATCC25177) with high accuracy. Through comparison with the genome sequences of H37Rv and CDC 1551, and re-sequencing of the corresponding mutation site in H37Rv, we identified 130 H37Ra-specific mutations which may lead to the phenotypic characters of H37Ra. Through analysis of these mutations and its effects to gene function, the mechanism of virulence attenuation in H37Ra was systematically studied.Second, we expressed and purified the mycobacterial MazG, and established the in vitro enzyme assay system. It was found that MtMazG was able to hydrolyze not only all canonical nucleoside triphosphates but also the non-canonical nucleotide, dUTP and 8-oxo-dGTP. suggesting a probable "house-cleaning" function proposed previously. We showed that, for the first time, MtMazG can hydrolyze abnormal NTPs. MtMazG was found to be a homotetramer and likely to have each subunit composed of a single core-domain flanked by two additional regions with unknown functions. This structure is distinct from that of the characterized MazG family proteins.In the third part, the effect of the A219E mutation in MtMazG activity was studied and its related mechanism was uncovered. It was found that the NTP-PPase activity was reduced to 7% of that of the wild-type enzyme under various conditions, suggesting that the A219E is a loss-of-function mutation. Because residue A141 of the EcMazG (corresponding to A219 of MtMazG) is located near the catalytic center motif for Mg2+ binding, we reasoned that the A219→E substitution of MtMazG might result in an increase of negative charge that alters Mg2+ binding. As revealed by the Mg2+ titration experiments, the MtMazG[A219E] exhibited a affinity for Mg2+ six times lower than that for MtMazG. The CD spectra of the MtMazG[A219E] enzyme exhibited significant alterations in the presence of Mg2+. These results suggest that the negative charge of the E219 side-chain induces rather drastic alterations in the secondary structures of MtMazG and this altered conformation may explain the severe deficiency of MtMazG[A219E] in hydrolysis.In the last part, we generated the M. smegmatis mazG null mutant by using the phage mediated specific tranducing strategy. The M. smegmatis mazG null mutant was not involved in starvation response, suggesting a function divergent from the E.culi MazG. It was found that deletion of mazG in M. smegmulis rendered the mycobacteria defective in response to oxidative stress. Intriguingly, under oxidative stress, both the mazG null and MtMazG[A219E]-expressing M. smegmatis strains failed to elevate relA, suggesting a specific role for the MazG NTP-PPase activity in oxidative stress-triggered, transcriptional activation of relA. The N-terniaml and C-termianl of MtMazG was found to be essential for NTP-PPase activity, while the corresponding mutation in E.coli MazG barely affects its NTP-PPase activity. Taken together, these results demonstrate that the MtMazG is structurally different from E. coli MazG.In summary, based on the findings of the comparative genomic study between H37Rv and H37Ra, this study elucidated the physiological role of MtMazG and the molecular mechanism of the loss-of-function MtMazG[A219E] mutation. Our study indicated that the Mtb MazG is noval NTP-PPase which is enzymatically. structurally and functionally divergent from E. coli MazG, and implicates a role for the MazG activity in the virulence of M. tuberculosis.