Browsing by Author "Steyn, Nastassja Lise"

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    Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis
    (Stellenbosch : Stellenbosch University, 2012-12) Steyn, Nastassja Lise; Gey van Pittius, N. C.; Warren, R. M.; Stellenbosch University. Faculty of Medicine and Health Sciences. Department of Biomedical Sciences.
    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.
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    Nucleoid gene regulation in mycobacteria
    (Stellenbosch : Stellenbosch University, 2017-12) Steyn, Nastassja Lise; Warren, Robin Mark; Gey van Pittius, Nico; Sampson, Samantha Leigh; Williams, Monique Joy; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains one of the leading causes of death worldwide. The ongoing tuberculosis epidemic and poor treatment outcomes can be attributed to the emergence of antibiotic resistant mycobacterial organisms as well as persistent M. tuberculosis infections. It is therefore imperative that we improve our understanding of M. tuberculosis pathogenesis to develop more effective anti-tuberculosis drugs and treatment strategies. M. tuberculosis is known to encounter adverse stress conditions such as nutrient starvation, hypoxia, nitrosative stress and low pH during infection and understanding how this pathogen adapts to its host environment is crucial in finding ways to circumvent tuberculosis disease. Conventional methods of investigating transcriptional responses, such as microarrays and ChIP-seq, are limited to the investigation of a single protein. Although valuable information has been obtained through this approach, adaptation to stress is likely mediated through several regulatory elements. Furthermore, new methodologies are required to identify these proteins. For example, nucleoid-associated proteins (NAPs), a group of global transcriptional regulators which have the unique ability to shape the bacterial chromosome, have been shown to be involved in the mycobacterial stress response. However, due to poor sequence homology with other bacterial organisms and conventional methodologies, only seven nucleoid associated proteins have been identified for mycobacterial organisms compared to 12 NAPs in Escherichia coli. Nucleoprotein - Mass Spectrometry (NP-MS) makes use of affinity purification of formaldehyde cross-linked RNA polymerase transcriptional complexes to identify proteins involved in chromosomal structure, and transcriptional and translational processes using mass spectrometry. This approach successfully identified various DNA and RNA associated proteins as well as numerous proteins associated with energy, carbon, lipid and amino acid metabolism. These results suggested that NP-MS was effective in isolating not only proteins directly involved in transcription and translation but also proteins associated with the RNA polymerase transcriptional complex and DNA. MSMEG_1060, MSMEG_2695, MSMEG_3754, MSMEG_4306 and MSMEG_5512 were identified as possible nucleic acid associated proteins through comparison of conserved proteins domains and were subjected to further investigation. Episomal expression of these proteins as FLAG-tagged fusion proteins in M. smegmatis revealed MSMEG_1060, MSMEG_2695, MSMEG_4306 and MSMEG_5512 to be putative DNA-associated proteins whilst no DNA association was found for MSMEG_3754. NP-MS was applied to investigate differences between DNA-associated proteins in exponential and stationary phase cultures. Investigation into the proteins which make up the RNA polymerase transcriptional complex and its associated proteins in stationary phase M. smegmatis cultures, revealed that NP-MS could effectively be used to identify proteins which are required for adaptation to stress in this organism. These included the dormancy response regulator DevR, the ribosome hibernation promoting factor (hpf), heat shock protein HspX and the universal stress proteins MSMEG_3811, MSMEG_3945 and MSMEG_3950. These results demonstrated the ability of the developed NP-MS method to identify the proteins which mediate DNA structure, and transcriptional and translational changes in M. smegmatis. Furthermore, we propose that NP-MS can be used to investigate the proteins associated with the RNA polymerase complex in not only mycobacterial species but also other bacterial organisms.