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ENGLISH ABSTRACT: Drug resistance in Mycobacterium tuberculosis is an increasing global problem. Drug resistance is mostly caused by single nucleotide polymorphisms (SNPs) within the bacterial genome. This observed increase in global incidence of drug resistant tuberculosis (TB) has sparked the search for new anti-TB drugs and the repurposing of drugs that are currently used against other organisms or species of mycobacteria. One such repurposed drug, clofazimine (CFZ), is currently used for the treatment of leprosy, caused by Mycobacterium leprae. The mechanism of action of CFZ is not clear, but it is hypothesized that CFZ is reduced by a mycobacterial type II NADH oxidoreductase (NDH-2). The reduction of CFZ drives the production of reactive oxygen species (ROS) which is toxic to the pathogen. The aim of this study was to elucidate the mechanism of CFZ resistance. Towards this aim, spontaneous in vitro CFZ resistant mutants were selected, characterized and whole genome was used identify SNPs which may cause CFZ resistance. Mutations were identified in a transcriptional regulator encoded by Rv0678, fatty-acid-AMP ligase, or FadD28 (Rv2941) and glycerol kinase or GlpK (Rv3696c). Mutations in Rv0678 have previously been shown to play a role in both CFZ resistance and bedaquiline (BDQ) cross-resistance, while no link has been found between CFZ resistance and mutations in fadD28 and glpK. The novel, non-synonymous SNP identified in Rv0678 resulted in the replacement of an alanine residue with threonine at codon 84, which is located in the DNA binding domain. Virtual modelling of the mutated Rv0678 protein showed that the A84T mutation may influence DNA binding, possibly due to its proximity to the DNA binding domain. This mutation caused a change in hydrophobicity, which may influence binding to DNA. Previous studies showed that mutations in Rv0678 resulted in the upregulation of mmpL5, a putative efflux pump. However, the mechanism whereby CFZ resistance occurs via increased abundance of this efflux pump in the cell wall is not clear and needs further investigation. The cross-resistance between CFZ and BDQ, caused by mutations in Rv0678, is of concern and may influence the planning of anti-TB drug regimens for the future. The roles of the other two mutations identified in this study in CFZ resistance is also not clear and requires further investigation. Finally, the findings of this study support the role of Rv0678 in CFZ resistance thereby suggesting that this gene could be useful as a diagnostic marker to test for CFZ resistance in clinical isolates.

AFRIKAANSE OPSOMMING: Middelweerstandigheid in Mycobacterium tuberculosis is 'n wêreldwye toenemende probleem. Middelweerstandigheid word meestal veroorsaak deur enkel nukleotied polimorfismes (SNPs) in die bakteriële genoom. Hierdie toename in middelweerstandige tuberkulose (TB) het gelei tot die soektog na nuwe anti-TB-middels en die alternatiewe aanwending van middels wat tans teen ander organismes of spesies van mikobakterieë gebruik word. Een so 'n alternatiewe…

Advisors/Committee Members: Victor, T. C., Paul, L. V., Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics..

▼ ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of the tuberculosis disease, is estimated to infect a third of the world’s population and is therefore, arguably, the most successful human pathogen in recorded history. Immense efforts to understand the genetic factors and biochemical processes underlying the complex interactions between M. tuberculosis and its host cells have delivered staggering insights into the profound proficiency by which this bacterium establishes and maintains an infection. It is now clear that M. tuberculosis can interfere with the immune responses initiated by host cells in such a manner as to subvert the various bactericidal conditions established by these cells and thus eliminate the tubercle bacilli that infect them. Specific characteristics of M. tuberculosis which provide it with this ability include a nearly impenetrable cell wall, secretion systems which secrete special factors which directly interact with host immune factors. This enables M. tuberculosis to modulate the activities of the host environment and unique metabolic adaptations of M. tuberculosis allows the organism to survive in the hypoxic, oxidative, nitrosative, acidic and nutrient poor environment of immune cell phagosomes and to persist for decades in a quiescent state in otherwise healthy people. New observations into the pathways which constitute energy, carbon and central nitrogen metabolism, among others, in M. tuberculosis, suggest that a carefully orchestrated homeostasis is maintained by the organism which may modulate the concentrations and ameliorate the effect of molecules that are important to defensive strategies employed by host cells. Here we discuss various recent studies as well as new information provided by this study, focusing on central metabolism and its regulation in M. tuberculosis. We aim to highlight the importance of nitrogen metabolism in the subversive response employed by M. tuberculosis to survive, colonise and persist in the host. We argue that the homeostatic regulation of nitrogen metabolism in M. tuberculosis presents a profound vulnerability in the pathogen which should be exploited with compounds that inhibit the activities of various effector proteins found in this pathway and that are unique to the organism. Such compounds may provide valuable novel chemotherapies to treat tuberculosis patients and may alleviate the burden of multiple drug resistance which plagues tuberculosis treatments. Specifically, in this study we investigate the role of M. bovis BCG glutamate dehydrogenase (GDH) and glutamate synthase (GltS) by subjecting knockout mutants of the aforementioned gene products to various cellular stress conditions. Furthermore, we investigated how the genomes of each M. bovis BCG strain was affected post deletion of the aforementioned protein products. The role of GDH was also tested in an murine macrophage model of infection to elucidate potential importance to colonisation and infection. This study provides novel results indicating an importance of GDH toward… Advisors/Committee Members: Wiid, Ian J. F., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics..

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ENGLISH ABSTRACT: The role of bacterial small RNA (sRNA), i.e. RNA species between 50-500bp in size, in virulence, pathogenesis and drug resistance is gaining interest. In some bacterial species, it had been shown to play a crucial role in bacterial transcriptional and post-transcriptional regulation. sRNAs from various pathogenic bacteria were shown to modulate bacterial responses to the host and environment. In Mycobacterium tuberculosis, the causative agent of tuberculosis, more than 1000 sRNA species have been identified already; but the role of these sRNA in pathogenesis, virulence and stress responses is not well studied. Central dogma suggests that drug resistance in M. tuberculosis is associated with mutations in specific genes. However, a number of clinical drug resistant isolates do not harbour mutations in these genes, implicating other factors such as unknown mutations, as well as altered regulation of these resistance genes. Prediction of resistance, using molecular methods, can therefore be inaccurate in cases where known mutations are absent. In cases where known drug-resistance associated mutations are absent, mutations in other genes that regulate such resistance-associated genes might influence drug resistance. Growing evidence, in other bacteria and M. tuberculosis, hints at a role for mutations in intergenic regions and sRNAs species to play a role in bacterial growth and drug sensitivity. In light of this we hypothesised that mutations in sequences encoding sRNA or in sRNA target sequences influence the phenotype of M. tuberculosis clinical isolates. Using previously identified sRNA genes; we screened a genomic bank of clinical M. tuberculosis isolates for the presence of mutations in these sRNA encoding genes. A large number of isolates showed mutations in genes encoding for sRNAs. Furthermore, over-expression of sRNA using the plasmid pMV306 in Mycobacterium smegmatis showed differences in growth indicating that the presence of the extra copies of the three sRNA (mcr3, ASpks and mpr6) had a phenotypic effect on the bacterium. Overexpressed sRNAs did not affect the bacterial drug resistance phenotypes, although this requires further investigation before concluding the effect of sRNAs on drug resistance. We successfully modified a method to extract and purify sRNAs from Mycobacterium species, clean enough to perform Real Time Polymerase Chain Reaction even with small amounts. However challenges were faced in terms of quantification. Another challenge that still remains is obtaining reference genes specifically for sRNAs as we currently have none.

AFRIKAANSE OPSOMMING: Die rol van klein ribonukleïnsure ( m.a.w RNS spesies van ongeveer 50-250bp in grootte) in bakteriële virulensie, patogenese en antibiotika weerstandigheid word al hoe meer bevraagteken. 'n Rol vir hierdie nukleinsure in transkripsie en post-transkripsie regulering was voorheen gewys in verskeie bakteriële studies, waar dit gedemonstreer was dat hierdie RNA spesies n rol speel vir die bakterieë om aan te pas in die gasheer se…

Advisors/Committee Members: Victor, T. C., Paul, Lynthia, Streicher, Elizabeth, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics..

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ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects one third of the world’s population and kills 1.7 million people per year. The increasing prevalence of multi- and extensively drug resistant M. tuberculosis strains means that there is an urgent need to develop new anti-TB drugs. The genome of M. tuberculosis has five copies of the ESAT-6 gene clusters (ESX-1, -2, -3, -4 and -5), which are essential for the survival (ESX-3) and pathogenicity (ESX-1 and ESX-5) of the bacterium. The ESX clusters encode for proteins which form a novel secretion system which has been shown to secreted small T-cell antigens of the esx gene family, as well as other proteins such as the PE and PPE’s. The mycosins are a family of genes situated in the ESX clusters which encode for putative subtilisin-like serine proteases. These proteins are the most conserved proteins within the five clusters. Apart from their conserved protein sequence, mycosin-3 is also an essential protein specific to the mycobacteria, which makes it an attractive potential drug target. Identifying the substrate(s) of mycosin-3 could help to understand the function of this enzyme and discover novel inhibitors from which new drugs could be designed. We hypothesize that the secreted products of the ESX system could be potential substrates for the mycosins. Specifically, we hypothesize that PE5, PPE4, esxG and esxH (all found in ESX-3) might be the substrates for mycosin-3. Mycosin-3, PE5, PPE4, esxG and esxH were thus cloned, expressed and purified respectively. The four substrates were used for protease assays using mycosin-3 as the protease. The protease-substrate mixture were subsequently separated on 2-D SDS-PAGE gels to check whether there were any cleavage of the four substrates. Although all the target fusion proteins were cloned and expressed successfully, the protease assay results showed no cleavage for any of the four substrates. Possible explanations for the failure of cleavage were: (1) impure enzyme and substrate(s); (2) inappropriate buffer conditions; (3) the hypothesized substrates might not be the substrates of mycosin-3; and (4) incorrect folding or modification of the target fusion proteins might have taken place. Future research will aim to address these possible limitations in order to fully elucidate the function and substrate specificity of mycosin-3 and to use this information for the design of novel drugs against M. tuberculosis.

AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat tuberkulose (TB) veroorsaak, infekteer `n derde van die wêreld se bevolking en veroorsaak die dood van 1.7 miljoen mense per jaar. Die verhoogde voorkoms van multi- en ekstensiewe middelweerstandige stamme van M. tuberculosis beteken dat daar `n ernstige nodigheid is om nuwe anti-TB middels te ontwikkel. Die genoom van M. tuberculosis het vyf kopieë van die ESAT-6 geengroepe (ESX-1, -2, -3, -4 en -5), wat essensieel is vir die oorlewing (ESX-3) en patogenisiteit (ESX-1 and…

Advisors/Committee Members: Gey van Pittius, Nicolaas Claudius, Warren, Robert Mark, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences..

▼ ENGLISH ABSTRACT: Central dogma suggests that mutations in target genes is the primary cause of resistance to first and second-line anti-TB drugs in Mycobacterium tuberculosis. However, it was previously reported that approximately 5% of Rifampicin mono-resistant clinical M. tuberculosis did not harbor mutations in the rpoB gene. The present study hypothesized that active efflux plays a contributory role in the level of intrinsic resistance to different anti-TB drugs (Isoniazid, Ethionamide, Pyrazinamide, Ethambutol, Ofloxacin, Moxifloxacin, Ciprofloxacin, Streptomycin, Amikacin and Capreomycin in RIF mono-resistant clinical M. tuberculosis isolates with a rpoB531 (Ser-Leu) mutation. This study aimed to define the role of Efflux pump inhibitors (verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine) in enhancing the susceptibility to different anti-TB drugs in the RIF mono-resistant clinical isolates. The isolates were characterized by determining the level of intrinsic resistance to structurally related/unrelated anti-TB drugs; determining the effect of EPIs on the level of intrinsic resistance in the isolates and comparing the synergistic properties of the combination of EPIs and anti-TB drugs. To achieve this, genetic characterization was done by PCR and DNA sequencing. Phenotyping was done by the MGIT 960 system EpiCenter software to determine the MICs of the different anti-TB drugs and the effect of verapamil and carbonylcyanide m-chlorophenylhydrazone on determined MICs. Due to inability to test reserpine in a MGIT, a different technique (broth microdilution) was used for the reserpine experiment. Additionally; fractional inhibitory concentrations (FIC) indices were calculated for each of these drugs. The FIC assess the anti-TB drugs/inhibitor interactions. STATISTICA Software: version 11 was used for statistical analysis. Results revealed that the RIF mono-resistant isolates were sensitive at the critical concentrations of all 10 drugs tested, with the exception of Pyrazinamide. This could be explained by the technical challenges of phenotypic Pyrazinamide testing. A significant growth inhibitory effect was observed between the combination of EPI and anti-TB drug exposure in vitro. This suggests that verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine play a significant role in restoring the susceptibility (decrease in intrinsic resistance level) of the RIF mono-resistant isolates to all anti-TB drugs under investigation. Additionally, a synergistic effect was observed by the combination treatment of the anti-TB drugs with the different EPIs. Based on these findings, we proposed a model suggesting that efflux pumps are activated by the presence of anti-TB drugs. The activated pumps extrude multiple or specific anti-TB drugs out of the cell, this in turn decrease the intracellular drug concentration, thereby causing resistance to various anti-TB drugs. In contrast, the addition of EPIs inhibits efflux pump activity, leading to an increase in the intracellular drug concentration and… Advisors/Committee Members: Victor, T. C., Louw, G. E., Warren, R. M., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Molecular Biology and Human Genetics..

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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.

AFRIKAANSE OPSOMMING: Multi-middel weerstandige tuberkulose (MDR-TB) word gedefinieer as weerstandigheid tot ten minste rifampisien (RIF)…

Advisors/Committee Members: 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: The current anti-TB drugs have had success in decreasing the number of deaths caused by TB, however, this success is limited by the emergence of drug resistant TB strains. Therefore, a novel TB therapy that limits the development of resistance has become necessary in an attempt to effectively control TB. The anti-TB drugs directly target mycobacterial enzymes, and potentiate the development of this resistance, and have therefore provided the rationale for this study. The aim was therefore to identify host macrophage genes that affect M. tb intracellular survival. The proposed alternative anti-TB therapy potentially involves the application of RNA interference (RNAi) and RNA activation (RNAa) biological processes that will target host genes, thereby inducing an indirect bactericidal effect. We hypothesized that macrophage genes that are differentially expressed by pathogenic and non-pathogenic mycobacterial species may be important in the regulation of M. tb intracellular survival. The lipid-rich mycobacterial cell wall is implicated in the excessive clumping of the mycobacterial cells in liquid culture. In order to minimize this, Tween 80 detergent was supplemented (mycobacteriaT). However, due to substantial evidence emphasising the detrimental effects of Tween 80 on the mycobacterial cell wall, mycobacteria were also cultured without Tween 80 (mycobacteriaNT), in order to investigate if the perturbed mycobacterial cell wall induced by Tween 80 affects the transcriptional response of macrophages. We endeavoured to develop a new method to culture mycobacteria without Tween 80 that will still generate single cells. We further hypothesized that the macrophage gene expression profile induced by mycobateriaNT differs from the response induced by mycobacteriaT. Differentiated THP-1 (dTHP-1) cells were infected with pathogenic and non-pathogenic mycobacteria (for 3 h, 24 h and 48 h with M. tb and M. bovis BCG, and 3 h and 8 h with M. smegmatis) cultured in the presence or absence of Tween 80. The expression of 12 macrophage genes, selected based on their involvement in the phagocytic pathway and autophagy, as well as their general involvement in the immune response, was determined by qRT-PCR and further analysed on the REST programme. The expression of each target gene was normalised relative to the expression of the reference gene (Beta actin). We observed that out of the 12 genes, TLR7 and VAMP7 were consistently downregulated in dTHP-1 cells infected with M. tbNT and upregulated in dTHP-1 cells infected with M. smegmatisNT. Their response to M. bovis BCG was inconsistent and not significantly different, and therefore could not be interpreted. Furthermore, CCL1 was upregulated by all the mycobacterial species. However, its expression was more pronounced in response to mycobacteriaNT, when compared to mycobacteriaT. Differential gene expression of TLR7 and VAMP7 in response to pathogenic and non-pathogenic mycobacteriaNT suggests that these 2 genes may be potential targets for RNAa-based anti-TB… Advisors/Committee Members: Baker, Bienyameen, Wiid, Ian, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Molecular Biology and Human Genetics..

▼ ENGLISH ABSTRACT: An important next step to Tuberculosis control relies on the translation of basic science and modern diagnostic techniques into primary health care clinics. These assays must be rapid, inexpensive, interpretation of results must be easy and they must be simple so that a healthcare worker with limited training can perform the tests under safe conditions. This study consists of four aims. The first aim was to develop a methodology to sterilize sputum specimens for rapid TB diagnosis and drug resistance testing. Candidate bactericides were identified from the literature, and tested for their bactericidal activity in Mycobacterium tuberculosis. We identified ultraseptin®aktiv as a powerful bactericidal agent which sterilizes sputum specimens for subsequent safe handling prior to light emitting diode microscopy and it also provides a DNA template for PCR-based tests. An algorithm has been proposed for the processing of specimens and rapid diagnosis of TB and drug resistant TB while patients wait for results. Recently, the World Health Organization has endorsed the MTBDRplus test for diagnosis of TB and drug resistant TB. However genotypic tests may have more problems than anticipated. With the HIV pandemic, an increase of non-tuberculous mycobacteria has been reported. The sensitivity of genotypic tests in specimens with underlying non-tuberculous mycobacterial species therefore requires further evaluation. This study therefore also aimed at determining the reliability of the MTBDRplus assay for detection of drug resistant TB where non-tuberculous bacterial load is high. Clinically relevant non-tuberculous mycobacterium DNA and DNA from a multi-drug resistant TB isolate were obtained. Ratios of the different NTM with the MDR-TB DNA were made and subjected to the MTBDRplus assay. Known mix NTM and TB infected clinical isolates and sputum sediments were also evaluated for TB and drug resistance detection on the MTBDRplus assay. Under these conditions, this study provides evidence that the MTBDRplus test cannot reliably detect TB and drug resistance TB in specimens with underlying non-tuberculous mycobacteria. Thirdly, to evaluate the sensitivity of the MTBDRplus assay for detecting drug resistance in hetero-resistant isolates, ratios were made using purified DNA from an MDR and pan-susceptible TB isolate. The MTBDRplus assay was then performed on the different ratios. We report that the MTBDRplus assay can efficiently detect wild type DNA in genes associated with resistance during the early evolution of drug resistance. However, in the later stage during treatment when both the wild type and mutants are present, the detection limit for the mutant DNA was 1:55. Due to these results, the MTBDRplus assay should still be further improved or other tests should be developed to address these limitations. And finally to combat cross amplicon contamination during the final steps of genotypic detection with the MTBDRplus assay, a proof of concept for a patentable closed tube line probe device was proposed on the… Advisors/Committee Members: Victor, Thomas C., Warren, Robin M., Louw, Gail E., University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Science. 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… Advisors/Committee Members: 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: Individuals latently infected with Mycobacterium tuberculosis (M.tb) contain the infection without showing signs of illness. Steroid hormones such as glucocorticoids (GCs) however can lead to reactivation of TB. Currently two different injectable contraceptives are available in South Africa, medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET). MPA is able to bind to and activate the glucocorticoid receptor (GR) and possesses selective GC activity, a pharmacological characteristic absent in NET. The aim of this study was to investigate the immune modulatory effects of the two contraceptives MPA and NET on immune responses to mycobacteria in vitro and in vivo. Human peripheral blood mononuclear cells (PBMCs) were infected with BCG (M. bovis Bacille Calmette-Guérin) and treated with MPA, NET, progesterone or cortisol and cytokine expression was measured in order to determine whether the synthetic progestins mimic endogenous progesterone or the GC cortisol. MPA, but not NET suppressed the expression of IFN-γ, IL-1α, IL-1β, IL-2, IL-12p40 and IL-13 similarly to cortisol. Furthermore expression of miRNAs, small double stranded RNA molecules that bind to complementary sequences in mRNAs of target genes and cause their degradation, was determined under the different experimental conditions. The expression of several miRNAs including miR-30c, miR-222, miR-454 and miR-331-3p were differentially influenced by MPA, cortisol and/or NET in PBMCs stimulated with BCG. For example, BCG induced the expression of miR-454 (p=0.003) which was then inhibited to basal levels by cortisol (p=0.008), MPA (p=0.002) and NET (p=0.002). These results demonstrate that steroid hormones including the contraceptives MPA and NET can modulate immune responses to mycobacteria at the miRNA level. To determine the effect of MPA and NET on BCG-induced expression of miRNAs in vivo a mouse model was used. C57BL/6 mice were injected weekly with either MPA or NET using a dose equivalent to humans and then infected with BCG. Mice treated with MPA had a higher spleen bacterial load 21 days after infection compared to both NET treated and control mice (p=0.023). In whole blood, MPA and NET treatment suppressed the BCG-induced production of miR-100 and miR-509-3p to basal levels. In contrast to NET, MPA induced expression of miR-99a expression independent of BCG infection. In the lung, the site of disease, a total number of 22 out of 377 miRNAs tested were differentially expressed 21 days after infection. The results of this study indicate that both synthetic progestins altered the immune response to BCG at the level of cytokine expression as well as the miRNA level. MPA was found to mimic cortisol by inhibiting secretion of inflammatory cytokines whereas NET appeared to have more progestogenic properties. Each of the steroid hormones was observed to induce a characteristic miRNA expression profile, both in vitro as well as in vivo, although not identical. These results highlight that the two contraceptives – although used… Advisors/Committee Members: Ronacher, Katharina, Walzl, Gerhard, Kleynhans, Leanie, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Molecular Biology and Human Genetics..

▼ ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg district, Cape Town, in the Western Cape, South Africa Background The evaluation of early tuberculosis (TB) treatment response is based on month 2 sputum culture status. This method of evaluation has a number of limitations: the test requires relatively advanced laboratory infrastructure and procedures, it takes several weeks to obtain results and is a relatively a poor marker at predicting treatment response. The discovery of potential host markers which reflect the efficacy of early treatment would be of great importance for clinical management of individual patients. The treatment failure would be detectable earlier than at week 8 of treatment. The duration of clinical trials of new anti-tuberculosis drugs may also be substantially reduced by such markers if these would be measurable earlier than at week 8 of therapy. Objectives 1) To evaluate diluted, 7-day whole blood cultures stimulated with live Mycobacterium tuberculosis (M.tb) for the presence of host markers of early TB treatment response 2) To evaluate an overnight, undiluted, M.tb antigen stimulated whole blood culture Quantiferon Gold In Tube (QFT-GIT) supernatants for host markers of early TB treatment response The study designs were as follows: In study one, baseline samples and samples from week 1, week 2 and week 4 of treatment from 30 cured TB patients were selected from a larger biomarker study, in which whole blood was stimulated with live M.tb or left unstimulated. Fifty seven host markers were measured in supernatants by multiplex cytokine arrays. In study two, baseline samples and samples from week 2 and week 8 of treatment from 19 cured TB patients were randomly selected from the placebo group in a micronutrient supplement study. QFT-GIT supernatants from these participants were assessed through multiplex cytokine arrays for levels of fifty seven host markers. All of the participants in both studies were Human Immunodeficiency Virus (HIV) negative. Changes in marker expression over time and between fast and slow responders to treatment were evaluated. Comparability between the two culture methods was assessed for markers that were evaluated in both studies. Results In study one, the majority of host markers showed significant changes over time in the unstimulated supernatants. Only GRO and IL-1beta changed significantly in an antigen-specific manner (background levels subtracted). No significant changes were observed between fast and slow responders. In study two, the majority of host markers showed significant changes over time in the unstimulated supernatants whereas only MDC and IL-4 changed during the observation period in antigen stimulated levels. Significant differences were observed between fast and slow responders at pre-treatment for IL-13 Ag-Nil and IL-1betaAg-Nil . Conclusion This study revealed, antigen-specific responses showed only limited potential for early TB treatment response monitoring, but may have potential in differentiating between… Advisors/Committee Members: Walzl, Gerhard, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences..

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ENGLISH ABSTRACT: BACKGROUND: The emergence of mycobacterium tuberculosis resistance to first line drugs has renewed interest in second-line anti-tuberculosis drugs. Generally, Paraaminosalicylic acid (PAS) is less potent and frequently more toxic than the first line drugs. Furthermore, the pharmacokinetics of PAS in children has not been well characterized. AIMS: The aims of the present study were (1) to determine the pharmacokinetics of PAS in pediatric patients, (2) to describe the discrepancy between children and adult pharmacokinetics and the appropriate dosing regimen of PAS and (3) to investigate the potential of the second-line anti-tuberculosis drugs PAS, terizidone and ethionamide (often used as first-line drug in children) to inhibit the catalytic activities of CYP450 1A2 and 2C9. PATIENTS: Twenty two patients with drug resistant tuberculosis were included in the study. Ten patients were children with mean age of 4.2 years (range: 1 to 12 years). Twelve patients were adults with mean age of 31.3 years (range: 18 to 53). 4 children (40%) and 4 adults (33.3%) were HIV positive and were on ART. METHODS: Children received 75 mg/kg twice daily on the first visit and after two weeks they received 150 mg/kg once. Adults received a standard 4 g twice daily. Blood samples were taken at different time points after the dose. In the additional study, the inhibitory effects of PAS, ethionamide and terizidone on phenacetin O-deethylation, a marker substrate of CYP1A2 and diclofenac 4’-hydroxylation, a marker substrate of CYP2C9, were studied using human liver microsomes. RESULTS: For the 75 mg/kg dose, the mean AUC was 233.3 =g•h/ml and the mean CL was 10.4 l/h/kg. The mean of the observed Cmax of the drug was 45.4 =g/ml and the mean Tmax was 4.8 hrs. For the 150 mg/kg dose, the mean AUC of PAS was 277.9 =g•h/ml and the mean CL was 47.1 l/h/kg. The mean of the observed Cmax of the drug was 56.5 =g/ml and the mean Tmax was 4.8 hrs. On the first visit the mean AUC was 368 =g•h/ml and the mean CL was 13.2 l/h/kg. The mean of the observed Cmax of PAS was 51.3 =g/ml and the mean Tmax was 5.2 hrs. On the second visit the mean AUC was 230 =g•h/ml and the mean CL was 23.9 l/h/kg. The mean of the observed Cmax of PAS was 37.6 =g/ml and the mean Tmax was 5.2 hrs. The comparisons between pharmacokinetics profile of PAS and patients characteristics e.g. age, indicated no statistically significant differences between children (both treatment regimens) and adult patients as well as HIV positive and negative patients. In the in vitro study, all drugs demonstrated no inhibition potency towards the investigated CYP450 enzymes. CONCLUSIONS:The dose of 75 mg/kg twice daily in children appears to be appropriate to achieve serum concentration above the PAS minimum inhibitory concentration of approximately 1 =g/ml. PAS, ethionamide and terizidone are unlikely to affect the metabolism of concomitantly administered medications that are metabolized by either CYP450 1A2 and/or 2C9 isoenzymes.

AFRIKAANSE…

Advisors/Committee Members: Bouic, Patrick, Stellenbosch University. Faculty of Health Sciences. Dept. of Medicine. Pharmacology..

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ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and causes approximately 2 million deaths per year. Extensive research has been done on this pathogen, however our knowledge of the mechanisms of pathogenicity remain limited. The M. tuberculosis genome contains five ESAT-6 gene cluster regions, ESX-1 to 5, which encode specialized type VII secretion systems. These secretion systems are known to secrete members of the ESAT-6/CFP-10 and PE/PPE protein families, some of which contribute to the pathogenicity and phagosomal escape of the pathogen. ESX-3 has been shown to be essential for in vitro growth and survival of M. tuberculosis. The expression of ESX-3 in M. tuberculosis is regulated by IdeR and Zur, in response to intracellular iron and zinc concentrations, respectively. Interestingly, ESX-3 is not essential for the growth and survival of the saprophytic organism M. smegmatis. In this study, we aimed to identify the subcellular localisation of the individual components of the ESX-3 secretion system in the non-pathogenic, fast-growing organism M. smegmatis. The esx conserved component (ecc) genes from ESX-3 were expressed from the episomal expression vector pDMNI as fusion proteins with green fluorescent protein (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) and MSMEG_0626 (eccE3) were successfully cloned into pDMNI and expression of fusion proteins was confirmed by Western blotting for MSMEG_0615-GFP, MSMEG_0616-GFP and MSMEG_0626-GFP in M. smegmatis. In the M. smegmatis ESX-3 knock-out (with MSMEG_0615 to MSMEG_0626 deleted) expression was confirmed for MSMEG_0615-GFP and MSMEG0626-GFP. Fluorescent microscopy determined that MSMEG_0615-GFP localised to a single mycobacterial pole in both strains. MSMEG_0616-GFP and MSMEG_0626-GFP were found to be membrane associated in M. smegmatis, while MSMEG_0626-GFP was found to be membrane associated in the M. smegmatis ESX-3 knock-out. The unipolar localisation of MSMEG_0615-GFP suggests that the assembled ESX-3 secretion system apparatus is situated at a single pole in M. smegmatis. Therefore, we hypothesize that MSMEG_0615 might act as a recruiter protein that is involved in the assembly of ESX-3 at the mycobacterial pole.

AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is ‘n patogene organisme wat ‘n derde van die wêreld se bevolking infekteer en eis jaarliks 2 miljoen lewens deur tuberkulose. Ten spyte van uitgebreide navorsing, is daar min kennis oor die meganismes van patogenisiteit van hierdie organisme. Die M. tuberculosis genoom bevat vyf duplikasies van die ESAT-6 geen groep gebiede, ESX-1 tot 5, wat kodeer vir gespesialiseerde Tipe VII sekresie sisteme. Hierdie sekresie sisteme is bekend vir die sekresie van lede van die ESAT-6/CFP-10 en PE/PPE proteïen families, waarvan sommige bydra tot die patogenisieit en fagosomale ontsnapping van hierdie organisme. ESX-3 is noodsaaklik vir die in vitro groei en oorlewing van M. tuberculosis. Die …

Advisors/Committee Members: Gey van Pittius, N. C., Warren, R. M., Stellenbosch University. Faculty of Medicine and Health Sciences. Department of Biomedical Sciences..

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ENGLISH ABSTRACT: Application of molecular fingerprinting highlights transmission as the driving force behind the drug resistant epidemic in South Africa. Different strains dominate within different geographical regions, which is a reflection of micro-epidemics of drug resistance in the different regions. Cluster analysis shows that strains within the same strain family are different. The Beijing drug resistant strain family is the most dominant strain family (31%) in the Western Cape and of particular concern is the highly transmissible Beijing cluster 220 strain in the Western Cape communities. This cluster is widespread in the region and was previously identified in a MDR outbreak in a high school in Cape Town. Results suggest that the spread of Beijing drug resistant cluster 220 in the community was due to a combination of acquisition of drug resistant markers and transmission. This study also indicate that atypical Beijing can acquire drug resistance and become fit amongst HIV infected individuals. This is contrary to believe that atypical Beijing strains are not frequently associated with drug resistance and are attenuated. This implies that HIV levels the playing field for all drug resistant strains. Mechanisms leading to the evolution of MDR-TB and XDR-TB in a mine setting with a wellfunctioning TB control program which exceeds the target for cure rates set by the WHO were investigated. Despite the excellent control program, an alarming increase in the number of drug resistant cases was observed in 2003 and subsequent years. Phylogenetic analysis shows sequential acquisition of resistance to first and second-line anti-TB drugs leading to the development of MDR and XDR-TB. Contact tracing indicate extensive transmission of drug resistant TB in the shafts, hospital and place of residence. This study shows that despite exceeding the WHO cure rate target, it was not possible to control the spread and amplification of drug resistance. In summary, as a top priority, future TB control plans need to address diagnostic delay more vigorously.

AFRIKAANSE OPSOMMING: Molukulêre tegnieke toon transmissie as die hoofrede vir die toename in die anti-tuberkulose middelweerstandigheid epidemie in Suid-Afrika. Die verskillende Mikobakterium tuberkulose rasse wat domineer in verskillende areas is ‘n refleksie van middelweerstandige mikro-epidemies in verskillende gebiede. Analise van identiese rasgroepe demonstreer dat ras families bestaan uit verskillende rasse. Die Beijing middelweerstandige rasfamilie is die mees dominante familie in die Wes-Kaap (31% van monsters van middelweerstandige families) en van spesifieke belang is die hoogs oordraagbare Beijing 220 groep. Hierdie groep is die mees wydverspreide groep in die studie area en was voorheen geïdentifiseer tydens ‘n meervoudige middelweerstandige uitbreking in ‘n hoërskool in Kaapstad. Die resultate dui aan dat die Beijing middelweerstandige groep 220 in die gemeenskap versprei as gevolg van ‘n kombinasie van middelweerstand verwerwing en…

Advisors/Committee Members: Victor, Tommie, Stellenbosch University. Faculty of Health Sciences. DST-NRF Centre of Excellence for Biomedical TB Research..

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ENGLISH ABSTRACT: Introduction: Tuberculosis (TB) treatment registers and laboratory records are essential recording and reporting tools in TB control programmes. Reliable data are essential for any TB control programme but under-registration of TB cases has been well documented internationally, due to under-reporting of patients on treatment or failure to initiate treatment. The accuracy and completeness of routinely collected data are seldom monitored. Aim: This study used record linking to assess the accuracy and completeness of TB treatment register data and the feasibility of estimating the completeness of bacteriological confirmed pulmonary TB registration in two high incident communities in South Africa with capturerecapture methods. Methods: All cases of bacteriologically confirmed TB defined as 2 smear-positive results and/or at least one culture-positive result were included. Record linking was performed between three data sources: (1) TB treatment registers; and (2) all smear and culture results from (a) the nearest central laboratory, and (b) the referral hospital laboratory. To estimate the completeness of TB treatment recording three-source log-linear capture-recapture models were used, with internal validity analysis. Results: The TB treatment registers had 435 TB cases recorded of which 204 (47%) were bacteriologically confirmed cases. An additional 39 cases that were recorded as nonbacteriological cases in the TB treatment register, were reclassified as bacteriologically confirmed. In addition, there were 63 bacteriologically confirmed cases identified from the laboratory databases which were not recorded in the TB treatment register. The final total number of bacteriologically confirmed TB cases across all 3 databases was 306, an increase of 50% over what had initially been recorded in the TB treatment register. The log-linear capture-recapture model estimated the number of bacteriologically confirmed TB cases not found in any of the data sources at 20, resulting in a total number of bacteriologically confirmed TB cases of 326 (95% CI 314-355). The completeness of registration of bacteriologically confirmed pulmonary TB cases was 79% after record linking and 75% after the capture-recapture estimate. Conclusions: The results presented in this thesis highlighted the concern regarding the accuracy and completeness of routinely collected TB recording and reporting data. A high percentage of bacteriologically confirmed cases from both laboratories were not recorded in the TB treatment registers. Capture-recapture can be useful, but not essential, for evaluation of TB control programmes, also in resource-limited settings, but methodology and results should be carefully assessed. The present study estimated the extent of the problem of underreporting of TB in South Africa and identified challenges in the process. Interventions to reduce underreporting of TB are urgently needed.

AFRIKAANSE OPSOMMING: Inleiding: Registers van tuberkulose (TB) behandeling en…

Advisors/Committee Members: Barnes, Jo, Beyers, Nulda, Stellenbosch University. Faculty of Health Sciences. Dept. of Interdisciplinary Health Sciences. Community Health..

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Bibliography

ENGLISH ABSTRACT: There is a progressive maturation of the immune system from infancy to adulthood. The immature immune system in early life is characterised by impaired macrophage function and antigen presentation as well as a higher naIve to memory T cell ratio with subsequent diminished IFN-y production. Children with tuberculosis often present with lymphadenopathy, the complications thereof or with systemic spread of the organisms. Adults generally manifest with pronounced systemic effects (such as weight loss and high fever) and immunopathology (such as cavitation and fibrosis). We hypothesised that the immunopathology in adults may be due to enhanced cytokine production in comparison to children. The first aim of this study was therefore to measure cytokine responses in healthy children and adults. Cytokine responses in patients with tuberculosis will be examined in future studies. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood obtained from 9 healthy children and 9 healthy adults. The cells were cultured in serum-free medium, unstimulated or polyclonally stimulated with Phytohaemagglutinin (PHA). Supernatants were harvested after which IFN-y, IL-2, TNF-a., IL-4 and IL-IO production was determined by means of ELISA analysis. Ri'J"A was ~ubsequently extracted from the cells followed by RT-PCR analysis for the semiquantitative determination of mRNA levels of these cytokines. PBMC isolated from healthy children produced significantly less IFN-y protein than adults. Futhermore, IFN-y production in the adults seemed to be trimodally distributed. No significant differences could be found in the production of IL-2, TNF-a, IL-4 and IL-] O. Although children produced low levels of IFN-y protein, their IFN-y, TNF-a, IL-2, IL-4 and IL-IO mRNA levels were comparable to that of adults. Tuberculosis is a major cause of mortality and morbidity, particularly in the third world. Ravensmead and Uitsig, two adjacent suburbs in the Western Cape, have a tuberculosis incidence of> I 000/100000 population. Also, up to 90 % of the children in the Western Cape have been reported to be infested by intestinal parasites such as Ascaris lumbricoides and Trichurius trichl/ria. Infection with M tuberculosis indut:es a Th 1 Stellenbosch University http://scholar.sun.ac.za iv In:.,c response, while intestinal parasites elicit a Th2 immune response. Th2 dominance induced by intestinal parasite infestations could predispose individuals to an enhanced susceptibility to M. tuberculosis. The second aim of this study was to investigate serum IgE levels, surrogate markers for Th2 activation, in the community. The serum 19B levels were subsequently correlated to the tuberculosis incidence per enumerator sub-district (ESD), crowding, female literacy and socio-economic levels. Similarly, the tuberculosis incidence per ESD was correlated with the above mentioned parameters. A significant positive correlation was found between tuberculosis incidence and the serum 19E levels in…

Advisors/Committee Members: Beyers, A. D., Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Biomedical Sciences..