The glutamate dehydrogenase of the slow growing mycobacteria : its function in nitrogen metabolism and importance to in vitro and intracellular survival

Viljoen, Albertus Johannes (2013-12)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

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.

AFRIKAANSE OPSOMMING: Die metabolisme van glutamiensuur in Mycobacterium tuberculosis, die oorsaaklike agent in die siekte tuberkulose, is in onlangse studies as ‘n belangrike faktor in die oorlewing van die bakterium in gasheer-mensmakrofaagselle geïmpliseer. Glutamien oksoglutaraat aminotransferase (GOGAT) en glutamiensuur dehidrogenase (GDH), die belangrikste ensieme wat onderskeidelik by die produksie en af-braak van glutamiensuur betrokke is, word deur hulle interaksie met glikogeen akkumulasie reguleerder (GarA) beheer, wat deur proteïen kinase G (PknG), ‘n virulensie faktor, gefosforileer word. Die rolle wat GOGAT en GDH in die oorlewing van M. tuberculosis in makrofaagselle vertolk is egter nog nie bestudeer nie. In hiérdie studie word die regulasie van GOGAT en GDH in respons op fluktuasies in stikstof beskikbaarheid in Mycobacterium bovis BCG, ‘n nabye familielid van M. tuberculosis, beskryf. Bewyse word voorgelê dat die aminosuur-residu wat deur PknG in GarA gefosforileer word, deur M. bovis BCG benodig word om in vitro te kan groei. Alhoewel die gene wat vir GOGAT en GDH in M. tuberculosis kodeer essensieël is, is M. bovis BCG mutante van die korresponderende gene suksesvol in hiérdie studie geskep. Daar word aangetoon dat GOGAT vir die de novo sintese van glutamiensuur benodig word, terwyl GDH noodsaaklik is vir die verbruik van die aminosuur as ‘n uitsluitlike stikstof-bron asook in die metabolisme van asparagien. Alhoewel groei van die GOGAT mutant in makrofaagselle nie aansienlik verskil het van wilde tipe M. bovis BCG nie, was die intrasellulêre groei van die GDH mutant verswak, wat daarop dui dat GDH ‘n belangrike funksie tydens infeksie van makrofaagselle verrig. Daar word verder aangetoon dat die intrasellulêre vereiste vir GDH aan funksies van dié ensiem onder suur-omstandighede of in die gebruik van glutamiensuur as ‘n enigste koostofbron gekoppel kan word. Die M. tuberculosis GDH is ‘n unieke ensiem, wat grootliks verskil van sy menshomoloog of die homoloë wat in die menslike ingewande flora voorkom en bied ‘n potensiële roete vir die ontwikkeling van nuwe chemoterapeutiese intervensie strategieë in die siekte tuberkulose.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/95470
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