M tuberculosis truncated hemoglobins in persistence

DESCRIPTION (provided by applicant): Recrudescence of latent tuberculous infection contributes significantly to the pathogenesis of disease caused by Mycobacterium tuberculosis (Mtb). The mechanisms by which the tubercle bacillus establishes latency and later reactivates are, however, poorly understood. Two truncated hemoglobins (trHb?s), HbN and HbO, encoded by the gene glbN and glbO, respectively, exist in Mtb. Initial characterization of HbN and HbO in M. bovis BCG has shown that: i) glbN and glbO are differentially expressed --the expression of HbN in vitro is most prominent in the stationary phase of growth, while HbO is invariably detected throughout the various growth phases; ii) both trHb?s have high affinity for oxygen, albeit via different mechanisms; iii) HbN and HbO can detoxify nitric oxide (NO) by conversion of the nitrogen oxide to nitrate; significantly, a BCG deletion mutant of glbN is markedly attenuated for its ability to consume NO; iv) the function of HbO may be essential. Based on these findings, we propose to test the hypothesis that HbN and HbO are required for the survival and/or persistence of Mtb within the host. This survival/persistence-promoting attribute can be due to the ability of HbN and HbO to detoxify the antimycobacterial NO. In addition, by virtue of their ability to bind oxygen with high affinity, HbN and HbO can function as an oxygen reservoir in the relatively anaerobic environment of the tuberculous granuloma, thereby optimizing the functions of critical intracellular oxygen-dependent enzymes. Finally, the ability of these trHb?s to avidly bind oxygen may protect Mtb against oxidative damage. To begin testing these hypotheses, we will take a genetic approach, by generating glbN and glbO mutants, to rigorously test the in vivo significance of Mtb trHb?s in survival and/or persistence using murine experimental TB models. We will also evaluate the roles of HbN and HbO in Mtb respiration and in protection against the adverse effects of NO on the respiratory process. Establishment of the significance of HbN and HbO in persistence and unraveling the biochemical and physiochemical properties of these hemeproteins will set the stage for developing novels anti-tuberculous agents effective against Mtb, particularly those in the dormant state.