Mycobacterium tuberculosis is a rod-shaped, acid-fast, human-specific pathogen and is the causative agent of the respiratory disease tuberculosis (TB). The bacterium is a significant source of morbidity and mortality worldwide, and is currently predicted to infect approximately 2 billion individuals. The success of M. tuberculosis as a pathogen is closely linked with its ability to establish latent infections in susceptible individuas and reactivate at later timed during periods of immunosuppression. Determinants important for the establishment, maintenance, and reactivation of M. tuberculosis from long-term, persistent infection within the host are poorly understood. It is thought that latency initiates following encasement of M. tuberculosis within granulomatous lesions, and the recognition of specific signals present within this environment that inhibit aerobic respiration and promote the transition of M. tuberculosis into an altered physiological state of nonreplicating persistence (NRP). Recently, a locus (Rv0081-Rv0088) predicted to encode determinants comprising a formate hydrogenlyase (FHL) were found to be upregulated following exposure of M. tuberculosis to conditions (hypoxia and nitric oxide) promoting NRP in vitro. In addition, this locus was found to be directly regulated by two response regulators (DosR/DevR and MprA) known to contribute to persistence by M. tuberculosis in vitro and in vivo. FHL plays an important role in energy metabolism within enteric bacteria during periods of microaerophilic/anaerobic growth by mediating the oxidation of formate to CO2 and H2 in the absence of an external electron acceptor. This proposal seeks to fill a current gap in our knowledge by investigating aspects of M. tuberculosis metabolism under physiologically relevant conditions. We hypothesize that M. tuberculosis synthesizes a functional FHL enzyme complex that is required for energy metabolism and survival during periods of NRP. To address this hypothesis, two specific aims have been proposed. First, the ability of M. tuberculosis FHL to mediate the oxidation of formate to CO2 and H2 will be examined using spectrophotometric- and gas chromatographic-based approaches. Second, determinants comprising the predicted FHL will be examined to determine if they interact to form FHL and are required for survival of M. tuberculosis during NRP. Collectively, these studies are expected to provide novel insights into M. tuberculosis physiology under conditions associated with NRP and latency. Delineation of the basis for formate metabolism may also identify new enzymes that can be targeted for therapeutic intervention of M. tuberculosis during periods of NRP when the bacterium is otherwise recalcitrant to currently available antibiotics.
Experiments described in this proposal will investigate the importance of FHL-mediate formate metabolism in the physiology of Mycobacterium tuberculosis during non-replicating persistence. Examination of determinants comprising this enzyme complex is expected to fill a critical gap in our knowledge of M. tuberculosis physiology by furthering our understanding of M. tuberculosis metabolism under conditions associated with persistent infection. Completion of this proposal may lead to identification of new enzymes that can be targeted for therapeutic intervention of M. tuberculosis during latency when the bacterium is otherwise recalcitrant to currently available antibiotics.
