Browsing by Author "Viljoen, Albertus Johannes"

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    The glutamate dehydrogenase of the slow growing mycobacteria : its function in nitrogen metabolism and importance to in vitro and intracellular survival
    (Stellenbosch : Stellenbosch University, 2013-12) Viljoen, Albertus Johannes; Wiid, Ian J. F.; Kirsten, Catriona J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
    ENGLISH ABSTRACT: Recent studies have implicated the metabolism of glutamate in Mycobacterium tuberculosis, the causative agent of tuberculosis disease, as an important factor in survival of the bacterium in the human host macrophage cells. Glutamine oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), the major enzymes involved in the production and break-down of glutamate respectively, are controlled through their interaction with glycogen accumulation regulator A (GarA) which is phosphorylated by protein kinase G (PknG), a determinant of virulence. However, GOGAT and GDH have not been investigated for their roles in the survival of M. tuberculosis in macrophage cells. In this study, the regulation of GOGAT and GDH in response to fluctuations in nitrogen availability is described in Mycobacterium bovis BCG, a close relative of M. tuberculosis. Evidence is presented that the amino acid residue of GarA which is phosphorylated by PknG is required for in vitro growth of M. bovis BCG. Although the genes encoding for GOGAT and GDH in M. tuberculosis are essential, M. bovis BCG mutants of the corresponding genes were generated successfully in this study. It is shown that GOGAT is required for de novo synthesis of glutamate, while GDH is required for utilization of the amino acid as a sole nitrogen source and in the metabolism of asparagine. While growth of the GOGAT mutant in macrophage cells was not appreciably different from wild type M. bovis BCG, intracellular growth of the GDH mutant was impaired, suggesting that GDH plays an important role during infection of macrophage cells. It is shown that the intracellular requirement for GDH may be linked to functions of this enzyme in acidic conditions or in the utilization of glutamate as a carbon source. The M. tuberculosis GDH is a unique enzyme which differs largely from its human homologue or the homologues found in the human gut flora and present a potential route for development of novel chemotherapeutic intervention strategies in tuberculosis disease.