Masters Degrees (Molecular Biology and Human Genetics)

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Browsing Masters Degrees (Molecular Biology and Human Genetics) by browse.metadata.advisor "Baker, Bienyameen"

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    Gene expression changes in macrophages infected with pathogenic M. tuberculosis and non-pathogenic M. smegmatis and M. bovis BCG
    (Stellenbosch : Stellenbosch University, 2014-04) Mpongoshe, Vuyiseka; Baker, Bienyameen; Wiid, Ian; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, 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 therapy, even though we could not conclude whether their response was specific to macrophages. In addition, the observed difference in the expression of CCL1 induced by mycobacteriaNT, compared to mycobacteriaT suggests that the perturbation caused by Tween 80 on the mycobacterial cell wall most likely affected the response of macrophages to infection with mycobacteria. Furthermore, this study has demonstrated a feasible method by filtration to generate single cells from mycobacteriaNT, which should be considered for future mycobacterial infection studies.
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    The host response to infection with pathogenic and non–pathogenic mycobacteria: a proteomics approach
    (Stellenbosch : Stellenbosch University, 2018-12) Mahlobo, Precious Zama; Baker, Bienyameen; Wiid, Ian; Leisching, Gina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Tuberculosis (TB) continues to be a major health problem worldwide. In 2017, 1.6 million TB associated deaths were reported (WHO, 2017). The etiological agent of TB disease is Mycobacterium tuberculosis (Mtb), and is a highly successful pathogen due to its ability to persist in the host. The immune system uses the non-specific innate immunity as the first line of defence against invading pathogens. The interplay between macrophages and mycobacteria is not yet fully understood. Mass spectrometry is one of the most effective tools for identification and quantitation of proteins from complex mixtures of biological samples. It has been shown that mycobacteria cultured in detergent medium and detergent-free medium induce differential macrophage host response. Following on a study that identified differentially expressed genes using high-throughput RNA sequencing, we aimed to identify and quantify protein expression of murine bone marrow derived macrophages infected with non-pathogenic mycobacteria, Mycobacterium smegmatis, Mycobacterium bovis BCG, and pathogenic mycobacteria, Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis R179 grown in a detergent-free medium. The differential proteomes of C57Bl/6 cells in response to Mtb infection, were analysed at 12 hours post infection using liquid-chromatography-tandem mass spectrometry (LC-MS/MS). Four proteins MYH9, TLN1, AHNAK and GAL-3 were expressed by pathogenic mycobacteria. Moreover, corresponding genes (Myh9, Tln1, Gal-3, and Ahnak) of the differentially expressed proteins were quantified by using quantitative PCR (qPCR) to monitor and analyse gene expression at later time points, 12, 24 & 96 hours post-infection. At the later time points, Myh9, Tln1 and Ahnak, were down-regulated indicating that these genes are only expressed at an early stage (up to 24 hours post-infection) of mycobacterial infection; while Lgal-3 was up-regulated by all slow growers (BCG, H37Rv & R179) at 96 hours post-infection. Galectin 3 is a binding protein known to control the survival of Mtb during infection. The significance of this protein can be further investigated in TB patients and healthy controls.
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    Mycobacterium tuberculosis, a major threat to health in South Africa : intracellular survival after treatment with novel drugs designed against the mycothiol pathway
    (Stellenbosch : University of Stellenbosch, 2010-12) Mazorodze, James Hove; Baker, Bienyameen; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences.
    ENGLISH ABSTRACT: Mycothiol (MSH) is unique to mycobacteria as the major low molecular weight cellular thiol responsible for protection of bacteria against oxidative stress. The design of drugs and inhibitors against enzymes of the mycothiol pathway was based on the premise that mycothiol is unique to mycobacteria, and is thus important for its survival. A total of 80 inhibitors designed against enzymes of the mycothiol pathway were screened for inhibition of growth on in vitro growing M. tuberculosis using the BACTEC 460TM assay. The most active compounds were further tested for inhibitory potential of M. tuberculosis within macrophages. Initial screening in the macrophage system was done using the human-like THP1 cell line and then mouse bone marrow-derived macrophages. In this investigation we established that phenothiazine can be exploited as an inhibitor of enzymes of the mycothiol pathway. Although tunicamycin significantly inhibited the growth of M. tuberculosis both in vitro and ex vivo; it was found to be cytotoxic to host macrophages. To this end we provide proof-of-concept that compounds which can inhibit the expression of mycothiol enzymes have potential as anti-tubercular drugs. The response of M. tuberculosis to stress conditions was assessed via LC-MS in which maximal levels of mycothiol were produced during the early time points of exposure to isoniazid. We used mycothiol-deficient (mshA) M. tuberculosis to investigate the role of mycothiol for survival as well as the resultant phenotype when such mutants are exposed to stress conditions. The mshA deletion mutants in M. tuberculosis were resistant to INH at concentrations which inhibited growth in the wild-type strains. We postulated that katG and inhA, the genes involved in INH metabolism, required mycothiol for their activation. Morphological alterations of M. tuberculosis within macrophages were assessed using electron microscopy approaches. In this way we attempted to follow the fate of M. tuberculosis within the phagosomes, and how mycobacteria is processed in phagosomes in terms of replication, survival and degradation. The establishment of a successful infection by M. tuberculosis depends on the initial encounter with host macrophages, which represent the first line of cellular defense against microbial invasion. At the interface between mycobacteria and macrophages, the complex outermost layer of the mycobacterial cell wall probably plays a role in facilitating host cell entry. Under normal conditions (i.e. ingestion of non pathogenic microorganisms), newly formed phagosomes intermingle contents and membrane with the successive compartments of the endocytic pathway (early endosomes, late endosomes, lysosomes) through a complex series of fusion and fission. As they are processed into phagolysosomes, they undergo gradual modifications by specific addition and removal of membrane constituents. In addition, they become acidified due to the vacuolar proton pump ATPase located in the membrane and acquire toxic constituents, including hydrolases that will ultimately destroy bacteria.
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    The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages
    (Stellenbosch : Stellenbosch University, 2021-03) Mkhonza, Mbali Nondumiso; Baker, Bienyameen; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Tuberculosis (TB) is a communicable disease caused by the virulent mycobacterial strain, Mycobacterium tuberculosis (M. tb). With an estimated 1.3 million deaths, of HIV uninfected individuals, and 10 million infected people in 2019, the World Health Organisation deemed TB a global epidemic and implemented the End TB Strategy to eradicate the disease by 2035. Since the current treatment regimen makes use of antibiotics, the increasing number of multi-drug resistant M. tb strains has resulted in a need for research into alternative options. One approach that could be of interest is host directed therapy which aims to identify and support the mechanism of action used by the host during its immune response to infection. Previously generated Ampliseq data identified CMPK2, which is suspected of playing a role in the differentiation of monocytic cells into macrophages, and RSAD2, associated with antiviral response, as being among the top upregulated genes following mycobacterial infection. Prior research on these genes had focused on their role during viral infections and thus, we aimed to better understand their involvement in the killing of mycobacteria in macrophages. During the present study, in vitro analysis of cultured THP-1 cells following differentiation into macrophages and infection with Mycobacterium smegmatis (non-pathogenic strain) and Mycobacterium bovis BCG (facultatively pathogenic strain) was performed. Thereafter, siRNA mediated knockdown in M. smegmatis was conducted to determine the effect that decreased expression of these genes had on the survival of the mycobacteria. For the vector-based knock up of RSAD2 and CMPK2, M. bovis BCG infected cells were treated with gene specific vector. Mycobacterial survival (via colony forming units) and relative gene expression (via qPCR) was then assessed. Gene expression results showed a significant difference (p=0.00030) between infected only and those treated with RSAD2 siRNA at 12 hours post M. smegmatis-infection, indicating that knockdown was achieved when using specific siRNA. Similarly, we showed that CMPK2 levels measured at 12 hours post M. smegmatis infection also achieved a significant decrease in the gene levels, p<0.0001. However, the mycobacterial survival results under these treatment conditions did not indicate a significant change following gene specific knockdown of CMPK2 or RSAD2, suggesting that the change in mRNA levels did not translate to an overall difference in the killing of the mycobacteria. Next, when a vector-based approach was used to knock-up CMPK2 and RSAD2, the results indicated no significant change in M. bovis BCG survival at 24 hours post infection. However, at 48 hours post M. bovis BCG infection, a significant decrease in the mycobacterial survival was observed following RSAD2 and CMPK2 vector knock up, p = 0.0003 and p= 0.0317 respectively. In conclusion, the present study demonstrated that successful siRNA-mediated knockdown of CMPK2 and RSAD2 in M. smegmatis could be achieved in infected cells on an mRNA level. Analysis of M. bovis BCG survival following knock-up with the specific gene vectors indicated a significant change at 48 hours post infection.