Browsing by Author "Willemse, Danicke"
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- ItemRegulation of efflux in rifampicin resistant mutants of Mycobacterium tuberculosis(Stellenbosch : Stellenbosch University, 2013-03) Willemse, Danicke; Williams, Monique Joy; Victor, Thomas Calldo; Warren, Robin Mark; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Multidrug resistant tuberculosis (MDR-TB), defined as having resistance to at least the first-line drugs, isoniazid and rifampicin (RIF), is a global health problem. Mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, are implicated in RIF resistance - with the S531L and H526Y mutations occurring most frequently. The level of RIF resistance varies for strains with identical rpoB mutations, which suggests that other factors play a role in RIF resistance. Efflux has been implicated in determining the intrinsic level of RIF resistance. Increased expression of the multidrug efflux pump, Rv1258c, following RIF exposure was observed in some Mycobacterium tuberculosis MDR clinical isolates and H37Rv RIF mono-resistant mutants, but not others. The factors influencing the induction of Rv1258c are poorly understood. The aim of this study was to investigate the effects of rpoB mutations on expression of Rv1258c and whiB7, a transcriptional regulator of Rv1258c, in M. tuberculosis H37Rv in vitro generated RIF resistant mutants, in the absence and presence of RIF. The promoter region of M. tuberculosis H37Rv Rv1258c was cloned into a position upstream of a lacZ gene (encoding β-galactosidase) in multi-copy episomal and integrating vectors. Vector functioning and the effect of rpoB mutations on Rv1258c promoter activity were initially investigated in the non-pathogenic related species, Mycobacterium smegmatis mc2155 rpoB mutants and subsequently in M. tuberculosis by doing β-galactosidase assays. qRT-PCR was done to investigate the effects of rpoB mutations on native Rv1258c and whiB7 gene expression. Episomal and integrating vectors were functional and the integrating vector system was used for subsequent β-galactosidase assays in M. tuberculosis. Rv1258c promoter activity in the S531L mutant was approximately 1.5 times less and in the H526Y mutant 1.5 times higher than that of the wild-type in M. smegmatis. Similarly, Rv1258c promoter activity in the S531L mutant was approximately half and in the H526Y mutant approximately double that of the wild-type in M. tuberculosis. A similar trend in Rv1258c and whiB7 expression to those observed using β-galactosidase assays were observed when investigating the native Rv1258c and whiB7 gene transcript levels compared to the wild-type using qRT-PCR, although differences were not significant. Exposure of the M. smegmatis and M. tuberculosis rpoB mutants to sub-inhibitory levels of RIF did not affect Rv1258c promoter activity. Mutations in rpoB had a marginal effect on Rv1258c and whiB7 transcript levels, but showed the same trend as that seen for Rv1258c promoter activity. It remains to be determined whether these differences are biologically significant. When considering efflux pumps as new targets for treatment, possible differences in efflux pumps expression due to different rpoB mutations should be considered.
- ItemRegulation of iron-sulphur cluster biogenesis in Mycobacterium tuberculosis(Stellenbosch : Stellenbosch University, 2016-12) Willemse, Danicke; Williams, Monique Joy; Warren, Robin Mark; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.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.
- ItemRv1460, a SufR homologue, is a repressor of the suf operon in mycobacterium tuberculosis(Public Library of Science, 2018) Willemse, Danicke; Weber, Brandon; Masino, Laura; Warren, Robin M.; Adinolfi, Salvatore; Pastore, Annalisa; Williams, Monique J.Iron–sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In Mycobacterium tuberculosis, the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 operon (suf operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, Rv1460, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the sufBCDS operon. Rv1460’s function in M. tuberculosis has however not been determined. In this study, we demonstrate that M. tuberculosis mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of Rv1460 and Rv1461 was observed in the mutant, implicating Rv1460 in the regulation of the suf operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in M. tuberculosis. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.