Browsing by Author "Marakalala, Mohlopheni Jackson"

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    Inhibition of a Mycothiol biosynthetic enzyme and a detoxification enzyme as anti-tubercular drug targets
    (2008) Marakalala, Mohlopheni Jackson; Steenkamp, DJ
    Includes abstract. Includes bibliographical references (leaves 131-141).
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    Investigation of mycobacterial cell wall genes and their requirement for survival in immune related stressful conditions
    (2020) Samuels, Veneshley; Marakalala, Mohlopheni Jackson; Ndlovu, Hlumani
    Tuberculosis (TB) disease, caused by the pathogen Mycobacterium tuberculosis (Mtb), remains a major global health problem claiming 1.5-2 million lives annually. One of the major factors contributing towards Mtb's success as a pathogen is its unique cell wall and its ability to counteract various arms of the host's immune response. Understanding these survival mechanisms will help us develop new therapeutic interventions that can enhance the capacity of the immune system to kill the pathogen. A recent genome scale study profiled a list of candidate genes that are predicted to be essential for Mtb survival of host mediated responses. One candidate was ftsEX, a protein complex comprised of an ATP binding domain, FtsE, and a transmembrane domain, FtsX. FtsEX functions through interaction with a periplasmic hydrolase, RipC. FtsEX homologs in other bacteria have been linked to a key role in regulation of PG hydrolysis during elongation and division. Using M. smegmatis as a model, we hypothesised that FtsEX and RipC are required in the regulation of PG hydrolysis during normal cell wall elongation and division under stressful conditions in vitro. Antibiotic sensitivity was confirmed using Alamar blue MIC determination assays, which showed that ftsEX and ripC had increased sensitivity to chloramphenicol and not to rifampicin, isoniazid and ethambutol. Our growth curve analysis showed that ftsEX and ripC are not essential for survival in normal growth conditions. However, ftsEX and ripC are conditionally essential for M. smegmatis in low salt media. Growth defects in this condition were characterized by short and bulgy cells, as well as elongated filamentous cells with visible chaining. Major morphological changes were seen under nitrosative stress. A higher proportion of cells struggled to divide normally and formed chains. Lateral branching was also observed in ΔftsE, ΔftsX and ΔftsEX but not in ΔripC. The protein complex was also required for survival in media containing rifampicin. Treatment with the drug exacerbated growth defects of all the mutants, which were much shorter than WT cells, indicating impairment in the elongation process. Collectively, mutants are much shorter in length with an exception of a few extremely lengthy cells, suggesting that ftsEX and ripC are required for both normal cell elongation and division and ultimately for survival in stressful conditions.
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    Investigation of the potential of Spleen Tyrosine Kinase (SYK) as a target for host-directed therapy during mycobacterial infection in macrophages
    (2022) Mohapi, Sephekana Samuel; Marakalala, Mohlopheni Jackson; Ndlovu, Hlumani
    Tuberculosis (TB) is a communicable disease caused by a single infectious agent, Mycobacterium tuberculosis (Mtb). TB affects mostly the lungs and despite treatment being available, it still causes long term functional disability due to collateral tissue damage. The TBburden is exacerbated by the lengthy treatment period of 6-12 months which may result in issues of toxicity and poor adherence. Novel therapeutics are therefore urgently needed. Host directed therapies (HDT) are currently a promising way forward for limiting tissue pathology caused by Mtb. Spleen tyrosine kinase (SYK) plays an important role in innate immune signalling. It is expressed on innate immune cells such as macrophages. Macrophages play a critical role in thepathophysiology of TB. They are the first responders to Mtb infection and are phagocytic cells that engulf and destroy Mtb. They also produce inflammatory cytokines such as TNF and IL- 1β. Recent studies have suggested an involvement of SYK in the inflammatory signalling cascade linked to necrotic and caseous regions of granulomas of TB patients. However, it is unclear what role SYK plays in the pathophysiology of TB and whether its inhibition would result in resolution of excessive tissue damage in the lungs. Our study is based on an in vitro infection model of Thp-1 derived macrophages. We differentiated Thp-1 monocytes into macrophages and infected them with BCG or the pathogenic laboratory strain Mtb H37Rv. We then treated infected macrophages with SYK inhibitors; Fostamatinib and Piceatennol, collected supernatants and analyzed cytokine production using enzyme-linked immunosorbent assay (ELISA). Moreover, we also evaluatedwhether SYK inhibition with Fostamatinib or Piceatennol might affect the intracellular survival of Mtb in macrophages. We also attempted to confirm the reduced expression of SYK at geneand protein level after treating infected cells with Fostamatinib. Our data showed that Fostamatinib reduced the production of inflammatory cytokines IL-6, IL- 1β and TNF- in macrophages infected with BCG. Similarly, these findings were also observedin macrophages that were infected with Mtb H37Rv, with the exception of IL-1β that was unaltered in macrophages treated with Fostamatinib. Moreover, Fostamatinib reduced the production of anti-inflammatory cytokine IL-10 and the chemokine monocyte chemotactic protein-1 (MCP-1) in macrophages infected with Mtb H37Rv. We observed that Fostamatinibrescued macrophages from cell death induced by both BCG and Mtb H37Rv. Finally, we showed that treatment with Fostamatinib also reduced bacterial loads inside macrophages. In essence, our study showed that SYK inhibition attenuate Mtb induced inflammatory profile in macrophages and aids in macrophage anti-mycobacterial effects. Further, it suggests that SYK inhibition might be an attractive avenue to explore further as a potential host-directed therapy for TB.
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    A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism.
    (International Union of Crystallography, 2012) Broadley, Simon Gareth; Gumbart, James Conrad; Weber, Brandon William; Marakalala, Mohlopheni Jackson; Steenkamp, Daniel Jacobus; Sewell, Bryan Trevor
    MshB, a zinc-based deacetylase, catalyses a step in the mycothiol biosynthetic pathway that involves the deacetylation of 1-O-(2-acetamido-2-deoxy--d-glucopyranosyl)-dmyo-inositol (GlcNAc-Ins), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy--d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) and acetate. In this study, MshB was expressed, purified and crystallized. A new crystal form was encountered in 0.1 M sodium acetate, 0.2 M ammonium sulfate, 25% PEG 4000 pH 4.6. The crystals diffracted to 1.95 A˚ resolution and the resulting electron-density map revealed glycerol and the reaction product, acetate, in the active site. These ligands enabled the natural substrate GlcNAc-Ins to be modelled in the active site with some certainty. One acetate O atom is hydrogen bonded to Tyr142 and is located 2.5 A˚ from the catalytic zinc. The other acetate O atom is located 2.7 A˚ from a carboxylate O atom of Asp15. This configuration strongly suggests that Asp15 acts both as a general base catalyst in the nucleophilic attack of water on the amide carbonyl C atom and in its protonated form acts as a general acid to protonate the amide N atom. The configuration of Tyr142 differs from that observed previously in crystal structures of MshB (PDB entries 1q74 and 1q7t) and its location provides direct structural support for recently published biochemical and mutational studies suggesting that this residue is involved in a conformational change on substrate binding and contributes to the oxyanion hole that stabilizes the tetrahedral intermediate.