Browsing by Author "Fang, Zhuo"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Elucidation of the structure and function of the Mycosins, a family of essential subtilisin-like serine proteases of Mycobacterium tuberculosis
    (Stellenbosch : Stellenbosch University, 2014-12) Fang, Zhuo; Gey van Pittius, Nicolaas Claudius; Schubert, Wolf-Dieter; 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 an ancient pathogen, which has been infecting humans for millennia. It remains globally spread, infecting one third of the world’s population and causing around two million fatal cases per year. The treatment of this infectious disease remains complex, time consuming, resource demanding and costly. In the absence of adherence, good quality drugs and appropriate treatment regimens, the pathogen is highly likely to develop resistance to the antibiotics used during treatment. Searching for new drug targets and developing new drugs are constantly in progress to address these issues. Type VII secretion system (T7SS) is a signature protein secretion system in mycobacteria, associated with virulence and nutrient acquisition. There are five copies of the T7SS in M. tuberculosis, namely ESX-1 to -5, with ESX-3 being essential for the bacterial growth in vitro. The importance of ESX-3 is supported by the fact that it influences two major iron uptake pathways in mycobacteria, namely mycobactin-mediated iron acquisition and heme acquisition. Mycosin-3 is the only membrane core component of ESX-3 that has a subtilisin-like serine protease signature and plays an essential role in ESX-3 secretion. Mycosin-3 has a unique catalytic specificity and its substrates are unknown. Elucidating the protein structure and determining the function of mycosin-3 will help the design of effective inhibitors to abolish the protease function providing for a potential therapeutic option. In this study, the mycosin-3 gene from the M. tuberculosis genome was cloned and expressed in Escherichia coli and the protein was purified in vitro, with the aim of conducting structural studies. However, the amount of soluble and stable mycosin-3 was insufficient to progress to X-ray crystallography for protein structure determination. According to the literature, this technical difficulty is not uncommon for M. tuberculosis genes because the gene transcription and protein production machineries in mycobacteria are distinct from the conventional protein production host E. coli. In vitro expression analysis suggested that mycosin-3 possibly exerts a toxic effect on the expression hosts: E. coli and Mycobacterium smegmatis. To overcome these complexities, M. smegmatis was used as a model organism for functional studies; the mycP3 gene was deleted from the genome, and the proteomes of the wild type and mycosin-3 deletion mutant were compared. No major phenotypic differences were observed between the wild type and mutant possibly because the model organism has an alternative exochelin-mediated iron acquisition pathway. Interestingly, in the absence of mycosin-3, one component of the mycobactin export system, MmpL5, was not detected in the whole cell lysate (containing both cytosolic and membrane fractions) by mass spectrometry although the mmpL5 gene was transcribed, suggesting rapid protein degradation. We hypothesize that mycosin-3 may plays a role in maintaining the integrity of the membrane protein MmpL5 prior to its secretion and in facilitating its localization on the membrane. The direct involvement of mycosin-3 in the posttranslational modification of MmpL5 is currently under investigation. This study provides evidence that mycosin-3 may be an attractive drug target - abolishing mycosin-3 could disable mycobactin export, with ensuring toxicity from intracellular mycobactin accumulation thereby eliminating M. tuberculosis.
  • Thumbnail Image
    Item
    Elucidation of the substrates of mycosin 3, an essential protease of Mycobacterium tuberculosis
    (Stellenbosch : University of Stellenbosch, 2011-03) Fang, Zhuo; Gey van Pittius, Nicolaas Claudius; Warren, Robert Mark; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences.
    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.
  • Thumbnail Image
    Item
    Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
    (Biomed Central, 2017-08) Fang, Zhuo; Newton-Foot, Mae; Sampson, Samantha Leigh; Gey Van Pittius, Nicolaas Claudius
    Background The ESX secretion system, also known as the Type VII secretion system, is mostly found in mycobacteria and plays important roles in nutrient acquisition and host pathogenicity. One of the five ESXs, ESX-3, is associated with mycobactin-mediated iron acquisition. Although the functions of some of the membrane-associated components of the ESX systems have been described, the role of by mycosin-3 remains elusive. The esx-3 gene cluster encoding ESX-3 in both Mycobacterium tuberculosis and Mycobacterium smegmatis has two promoters, suggesting the presence of two transcriptional units. Previous studies indicated that the two promoters only showed a difference in response under acid stress (pH 4.2). This study aimed to study the effect of a mycosin-3 deletion on the physiology of M. smegmatis and to assess the promoter activities in wildtype, mycosin-3 mutant and complementation strains. Results The gene mycP 3 was deleted from wildtype M. smegmatis via homologous recombination. The mycP 3 gene was complemented in the deletion mutant using each of the two intrinsic promoters from the M. smegmatis esx-3 gene cluster. The four strains were compared in term of bacterial growth and intracellular iron content. The two promoter activities were assessed under iron-rich, iron-deprived and iron-rescued conditions by assessing the mycP 3 expression level. Although the mycP 3 gene deletion did not significantly impact bacterial growth or intracellular iron levels in comparison to the wild-type and complemented strains, the two esx-3 promoters were shown to respond inversely to iron deprivation and iron rescue. Conclusion This finding correlates with the previously published data that the first promoter upstream of msmeg0615, is upregulated under low iron levels but downregulated under high iron levels. In addition, the second promoter, upstream of msmeg0620, behaves in an inverse fashion to the first promoter implying that the genes downstream may have additional roles when the iron levels are high.