Thesis (MScMedSc)--University of Stellenbosch, 2011. / 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 ESX-5) van die bakterium. Die ESX
groepe enkodeer vir proteïene wat `n nuwe uitskeidingssisteem vorm wat bewys is om klein T-sel
antigene van die esx geenfamilie, sowel as ander proteïene soos die PE en PPE proteïene uit te skei.
Die mycosins is `n familie gene wat in die ESX geengroepe voorkom en wat waarskynlik enkodeer
vir subtilisin-agtige serine proteases. Hierdie proteïene is die mees gekonserveerde proteïene in die
vyf geengroepe. Mycosin-3 is `n essensiële protein wat spesifiek in die mikobakteriëe voorkom,
sodat dit `n aantreklike teiken vir die ontwikkeling van middels is. Die identifisering van die
substrate van mycosin-3 kan moontlik help om die funksie van die ensiem te verstaan en om nuwe
inhibeerders vir die ensiem te ontdek, wat kan lei tot die onwikkeling van nuwe middels. Ons
hipotese is dat die uitgeskeide proteïene van die ESX sisteem moontlik die substrate van die
mycosin proteïene kan wees. Meer spesifiek, ons hipnotiseer dat die proteïene PE5, PPE4, esxG en
esxH (wat almal in ESX-3 voorkom) die substrate vir mycosin-3 kan wees. Mycosin-3, PE5, PPE4,
esxG en esxH is afsonderlik gekloneer, uitgedruk en gesuiwer. Die vier substrate is gebruik vir
protease proewe met mycosin-3 as die protease. Die protease-substraat mengsel is hierna deur
middel van 2-D SDS-PAGE geanaliseer om te kyk of daar enige kliewing van die vier substrate
voorgekom het. Alhoewel al die teiken fusieproteïene suksesvol gekloneer, uitgedruk en gesuiwer is,
het die protease proewe geen kliewing getoon vir enige van die vier potensiële substrate nie.
Moontlike verklarings vir hierdie negatiewe resultaat is die volgende: (1) ensiem en substrate was
moontlik onsuiwer; (2) bufferkondisies was moontlik nie korrek nie; (3) gehipotiseerde substrate
mag moontlik nie substrate van mycosin-3 wees nie; en (4) nie-korrekte vouing of modifisering van
die teiken proteïene kon moontlik plaasgevind het. Toekomstige navorsing sal daarop gemik wees
om hierdie beperkinge aan te spreek om sodoende die funksie en substrate van mycosin-3 te kan
ontdek en nuwe middels teen M. tuberculosis te ontwerp.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6752 |
| Date | 03 1900 |
| Creators | Fang, Zhuo |
| Contributors | Gey van Pittius, Nicolaas Claudius, Warren, Robert Mark, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xiv, 81 p. : ill. |
| Rights | University of Stellenbosch |
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