This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mycobacterium tuberculosis (Mtb) accounts for more than 1.5 million deaths worldwide yearly. Skin test reactivity indicates that approximately two billion people in the world are infected with Mtb. AIDS patients are especially susceptible and the impact of tuberculosis (TB)-HIV is greater in the developing world. Even individuals that control the primary infection have the risk of developing active TB since the elimination of infecting bacilli is not complete. This is due to the fact that Mtb can maintain redox homeostasis in the adverse conditions of the granuloma and it can also resist the host stress response mediated by the reactive oxygen and nitrogen intermediates. Thus, viable but non-replicating or latent bacilli still remain lurking in the host, a condition denominated latent TB. Latent TB is one of the main obstacles to disease eradication in developed countries and a problem yet to come of age in the developing world, currently overwhelmed by the high number of primary TB cases. Little is known about the molecular mechanisms leading to TB latency and reactivation. However, research has determined that the maintenance of Mtb latency involves: (1) survival in an anaerobic and reducing environment by controlling redox homeostasis at the reducing nucleotide pool (e.g., extracellular bacilli in the interior of the granuloma), (2) survival under oxidative stress by controlling RNI and ROI levels (e.g., intracellular bacilli in phagocytic cells), (3) peptidoglycan and DNA repair by non-redox mechanisms (extracellular phase), and (4) protein oxidative damage repair by controlling redox homeostasis at the sulfur metabolic pool (intracellular phase). The esponse of Mtb to redox stress plays a key role in the maintenance of latency and subsequent reactivation. In this context, we propose the hypothesis that the nicotinamide nucleotide and sulfur pools control redox homeostasis in tuberculosis latency. To test this hypothesis, we propose to: 1) Perform 1D and 2D metabolomic and metabolomic flux studies comparing Mtb wild type and single/double mutant strains (e.g., ald, cbs) potentially impaired in latency. 2) Compare the susceptibility of Mtb wild type and mutant strains to the macrophage reactive oxygen/nitrogen intermediate (ROI, RNI) stress response. 3) Test/synthesize potential inhibitors of the enzymes that control redox homeostasis in latent TB and test their activity in the in vitro Wayne model of TB latency. The significance of the studies proposed is that we will use a novel methodology coupled with molecular genetic analysis to analyze the dynamic equilibrium established in Mtb latency, a key aspect of pathogenesis. We expect these studies will lead to the development of novel inhibitors capable of providing an effective control of latent tuberculosis.
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