Regulation of iron-sulphur cluster biogenesis in Mycobacterium tuberculosis

Willemse, Danicke (2016-12)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Iron-sulphur (Fe-S) clusters are protein cofactors that are important for the functioning of many proteins involved in diverse processes in Mycobacterium tuberculosis. Complex Fe-S cluster biogenesis systems are required for their synthesis, to protect the clusters from the deleterious effects of reactive oxygen species in vivo. The suf system is the primary Fe-S cluster biogenesis system in M. tuberculosis and the components are encoded in an operon consisting of seven genes (Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466). The first gene in the operon, Rv1460, is predicted to encode a transcriptional regulator based on homology with the cyanobacterial suf operon repressor, SufR. This study aimed to determine whether Rv1460 is involved in the regulation of suf operon expression and M. tuberculosis physiology. In order to address this knowledge gap, attempts were made to generate three distinct Rv1460 deletion mutants in M. tuberculosis H37Rv. Surprisingly, generation of only one of the mutants, Rv1460stop, in which Rv1460 is truncated by insertion of a premature stop codon, was successful. This suggests that Rv1460 is not essential for the in vitro growth of M. tuberculosis. Our inability to generate the ΔRv1460 and Rv1460ΔDNAbd mutants may be due to polar effects on the expression of downstream genes, which make these mutants non-viable. Analysis of the Rv1460stop mutant’s growth revealed that Rv1460 is required for normal growth on solid and in liquid media, under standard culture conditions. The Rv1460stop mutant was more sensitive to ROS, indicating the importance of Rv1460 in oxidative stress response, and potentially implicating it in intracellular survival and pathogenesis. The Rv1460stop mutant’s growth was, surprisingly, not impaired under iron limitation. Gene expression studies done on the wild-type, Rv1460stop mutant and complementation strain revealed that Rv1460 acts as a transcriptional repressor of itself and the rest of the suf operon, since transcript levels of both Rv1460 and Rv1461 increased in the Rv1460stop mutant. Rv1460 was shown to be co-transcribed with the rest of the operon. Transcript levels, however, also suggested that Rv1460 may be independently transcribed from the rest of the gene cluster. Electrophoretic mobility shift assays done with recombinant Rv1460 demonstrated binding of Rv1460 to the Rv1460 promoter and within Rv1461. This indicates that Rv1460 mediates transcriptional control through direct interaction with the suf operon DNA. Recombinant Rv1460 was shown to form dimers in solution and to coordinate an Fe-S cluster in vitro, which has important implications for its function as a regulator, because the affinity of Fe-S cluster containing regulators for DNA is often influenced by the presence and redox state of their cluster. The role of three conserved cysteine residues (C203, C216, C244), predicted to be involved in Fe-S cluster coordination in Rv1460, could not be confirmed, as replacing these residues with serine residues did not alter their ability to coordinate an Fe-S cluster. The regulation of the suf operon in M. tuberculosis is multi-faceted, probably because Fe-S cluster biogenesis needs to be fine-tuned to allow survival within the host. This study indicates that Rv1460 plays a key role in this regulation and in M. tuberculosis physiology.

AFRIKAANSE OPSOMMING: Yster-swawel (Fe-S) groepe is protein ko-faktore wat belangrik is vir die funksionering van baie proteïne betrokke in ‘n wye verskeidenheid prosesse in Mycobacterium tuberculosis. Omdat dit sensitief is vir reaktiewe suurstof groepe in die sel, word komplekse Fe-S sintese sisteme vir die sintese van Fe-S groepe benodig. Die suf sisteem is die hoof Fe-S sintese sisteem in M. tuberculosis en word deur ‘n operon wat uit sewe gene bestaan (Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466) enkodeer. Die eerste geen in die operon, Rv1460, enkodeer ‘n transkripsionele reguleerder gebaseer op homologie met SufR, ‘n suf sisteem repressor in cyanobacteria. Die doel van hierdie studie is om te bepaal of Rv1460 by die regulering van die suf operon se uitdrukking en die fisiologie van M. tuberculosis betrokke is. Om hierdie kennis gaping aan te spreek, is daar gepoog om drie mutante, waarin dele van Rv1460 weggelaat is, in M. tuberculosis H37Rv te genereer. Verbasend kon net een van die mutante, Rv1460stop, waarin die proteïen as gevolg van ‘n vroegtydige stop kodon nie gemaak word nie, gegenereer word. Die groei van die Rv1460stop mutant is negatief beïnvloed. Dit is moontlik dat die effek van die weglatings in die ander twee mutante, ΔRv1460 en Rv1460ΔDNAbd, die uitdrukking van die daaropvolgende gene negatief beïnvloed en veroorsaak het dat die mutante nie gegenereer kon word nie. Die Rv1460stop mutant was meer sensitief vir reaktiewe suurstof groepe, wat impliseer dat Rv1460 belangrik is vir die intrasellulêre groei en patogenese van M. tuberculosis. Geen uitdrukking studies het getoon dat Rv1460 die hele suf operon se uitdrukking onderdruk omdat transkripsie van Rv1460 en Rv1461 toegeneem het in die Rv1460stop mutant in vergelyking met die H37Rv stamvader. Variasie in die transkripsie vlakke van Rv1460 en Rv1461, kan daarop dui dat Rv1460 appart van die res van die gene getranskribeer kan word. Dit dui daarop dat daar ‘n promoter voor die Rv1461 geen kan voorkom. Elektroforetiese mobiliteits verskuiwing assays is met gesuiwerde Rv1460 proteïen gedoen en het getoon dat Rv1460 proteïen instaat is om aan die Rv1460 promoter sowel as binne-in die Rv1461 geen te bind. Dit wys dat Rv1460 die gene direk reguleer deur aan die DNA te bind. Gesuiwerde Rv1460 proteïen form dimere in oplossing en koördineer ‘n Fe-S groep. Dit het belangrike implikasies vir die funksionering van Rv1460 as ‘n reguleerde, omdat die affiniteit van ander reguleerders vir DNA beïnvloed word deur die Fe-S groep wat dit koördineer. Die Fe-S groep van Rv1460 fasiliteer waarskynlik die suf operon se reaksie op interne Fe-S groep aanvraag. Die rol van die drie sisteïen residue (C203, C216, C244) in Rv1460 se koördinering van ‘n Fe-S groep is steeds onbekend omdat die Rv1460 proteïen variante wat gemuteerde sisteïen residue bevat steeds in staat was om die Fe-S groep te koördineer. Dit is nie verbasend dat die regulering van die suf sisteem in M. tuberculosis kompleks is aangesien die uitdrukking van die suf sisteem fyn gereguleer moet word om oorlewing in die gasheer te verseker. Hierdie studie dui daarop dat Rv1460 vir die regulasie van die suf sisteem en die fisiologie van M. tuberculosis belangrik is.

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