Browsing by Author "Du Plessis, Juanelle"
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- ItemClinically relevant mutations contributing to drug resistance in Mycobacterium tuberculosis(Stellenbosch : Stellenbosch University, 2017-12) Du Plessis, Juanelle; Sampson, Samantha Leigh; Wigneshweraraj, Sivaramesh; Warren, Robin Mark; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Single nucleotide variants are the underlying driver of drug resistance, strain fitness and adaptation in Mycobacterium tuberculosis and investigating the mechanistic and physiological aspects of these mutations is key to our understanding of the biology of this pathogen. Rifampicin, one of the most powerful first line drugs used to treat tuberculosis, inhibits transcription in M. tuberculosis by binding to the β subunit of RNA polymerase (RNAP). However, mycobacteria are able to evade binding of this drug by acquiring mutations in the rpoB gene. These resistance-conferring mutations are essential for the survival of M. tuberculosis in the presence of rifampicin, however they impart a fitness cost to the bacterium due to a presumed reduction in transcription efficiency and subsequent changes in gene expression. One of the mechanisms M. tuberculosis uses to buffer the effects of this fitness cost is to acquire compensatory mutations in rpoC and rpoA. As RNAP is at the core of all mechanisms of gene regulation in M. tuberculosis, it is not surprising that mutations within this enzyme led to pleiotropic effects. As this remains a poorly understood area of mycobacterial physiology, the current work encompasses a triad of studies which aims to better understand functional aspects of M. tuberculosis RNAP, and the role of rpoB and rpoC mutations in drug resistance. First, the effect of a bacteriophage protein, Gp2, was investigated to determine whether it is able to inhibit RNAP in M. tuberculosis. As Gp2 is known to bind to the β’ subunit of RNAP in Escherichia coli, positive findings from this work would provide a framework for the identification of novel compounds that inhibit transcription in the presence of rpoB mutations, affecting the β subunit. By way of in vitro and in silico analysis, it was found that Gp2 binds to and inhibits RNAP in M. tuberculosis, however to a much lesser degree than it does in its de facto host, E. coli. Nonetheless, future studies can build on our findings as in silico modification of Gp2 could identify a structure which allows for stronger binding affinity of the protein. Secondly, the effect of rpoB and rpoC mutations on the function of mycobacterial RNAP was investigated using a series of in vitro transcription assays. Radioactivity-based assays were performed using purified wildtype and mutant versions of the RNAP complex, to assess enzyme activity and promoter affinity. Furthermore, the use of a fluorescence-based assay was trialled to develop a comparable method without the use of radiolabelled nucleotides. Lastly, we undertook a study to understand the effect of rpoC mutations on the transcriptome of M. tuberculosis. For this purpose, serial clinical isolates were selected where the acquisition of an rpoC mutation was observed. These samples were used for whole genome sequencing and gene expression analysis, which revealed a potential link between the rpoC V483G mutation and upregulation of Rv2416c (eis) and Rv1258c (tap). Serendipitously, genomic data also revealed that an ald mutation was acquired alongside the rpoC mutation. Recently, ald has been described as a novel gene linked to D-cycloserine resistance in M. tuberculosis, however, to date, the mechanism of drug resistance has not been determined. Given the unique opportunity to study the effect of ald mutations on gene expression in our study, we investigated two genes which were found to be differentially expressed in a clinical isolate with an ald mutation. A turbidity-based microdilution assay revealed that upregulation of Rv0577, a putative glyoxalase, led to an increase in the minimum inhibitory concentration of D-cycloserine, a finding which provides novel insight into the mechanism of D-cycloserine resistance in M. tuberculosis. In summary, this body of work has contributed to existing knowledge surrounding drug resistance and compensatory adaptation in M. tuberculosis.
- ItemDeciphering the impact of rpoB mutations on the gene expression profile of Mycobacterium tuberculosis(Stellenbosch : Stellenbosch University, 2014-04) Du Plessis, Juanelle; Victor, Thomas Calldo; Warren, Robin Mark; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Mycobacterium tuberculosis is the etiological agent for tuberculosis, an infectious disease which is one of the leading causes of morbidity and mortality in developing countries. The emergence of drug resistant tuberculosis has negatively impacted the efficacy of current treatment regimens and threatens to undermine tuberculosis control programs worldwide. Rifampicin forms the backbone of the World Health Organization’s recommended treatment regimen for the treatment of drug susceptible tuberculosis. Resistance to rifampicin is caused by mutations in the 81 bp core region of the rpoB gene which encodes the β subunit of RNA polymerase. Numerous studies have shown that mutations at codons 531 and 526 are the most frequent in clinical isolates, yet little is known concerning the mechanistic effect of these mutations on the fidelity of RNA polymerase. In the present study, we aimed to determine the influence of rpoB mutations on the gene expression profile of M. tuberculosis cultured in vitro. To accomplish this, rifampicin resistant clinical isolates and spontaneous mutants (selected in vitro from H37Rv and a drug-sensitive clinical strain) harbouring rpoB H526Y and S531L mutations were subjected to whole genome sequencing and genome-wide transcriptional profiling. When comparing the transcription profile of H37Rv to the in vitro rpoB mutants, a large proportion of the differentially expressed genes were found to encode for proteins involved in intermediary metabolism and respiration; and cell wall and cell processes. The majority of these differentially expressed genes were downregulated. Prominent differential expression in the same functional categories was also evident when comparing the clinical isolates with these mutations; however, a greater number of genes were differentially expressed in this case. Furthermore, expression of genes that are part of the WhiB7 regulon were found to be upregulated in the rpoB526 mutants, and downregulated in the rpoB531 mutants. These findings indicate that both the position of the rpoB mutation, as well as the genetic background of the strain, play an important role in the gene expression profile of rpoB mutants. Surprisingly, transcriptional profiling of cultures that were exposed to the critical concentration of rifampicin for 24 hours did not exhibit significant differential gene expression. Whole genome sequencing, followed by bioinformatic analysis, revealed that the in vitro mutants harbour synonymous and non-synonymous single nucleotide polymorphisms in addition to the respective rpoB mutations. This suggests that the mycobacterial genome is constantly evolving, challenging previous assumptions of relatively static mycobacterial genomes. The findings from this research have provided novel insight into understanding the influence of resistance-conferring mutations on the biology of M. tuberculosis and have shown that further studies are urgently needed to better understand the complex physiology of this pathogen. This knowledge will be critical for the success of future drug development endeavours.