Thesis (MScMedSc (Biomedical Sciences. Human Biology and Human Genetics))--University of Stellenbosch, 2010. / Thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Medical Biochemistry at the Faculty of Health Sciences, University of Stellenbosch. / ENGLISH ABSTRACT: Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease which causes
approximately 2 million deaths each year. Despite extensive research on tuberculosis and M.
tuberculosis, little is understood of the mechanisms of pathogenicity of the organism. The genome of
M. tuberculosis contains five ESAT-6 gene cluster regions, each of which contains genes encoding
proteins involved in the formation of a dedicated protein secretion system. Included in these regions
are genes encoding exported T-cell antigens, serine proteases, ATP-binding proteins and other
membrane-associated proteins. Although it is known that some of these secretion systems are
involved in virulence and phagosomal escape of M. tuberculosis, and that deletion thereof causes
attenuation of the organism, the structure, substrates and functions of the systems are largely
unknown. Understanding the structure of the ESX secretion systems will advance our understanding
of the mechanisms of mycobacterial pathogenicity and provide clues to ways in which to interfere with
these virulence mechanisms.
The ESAT-6 gene cluster region 3, encoding the ESX-3 secretion machinery, is the only ESAT-6 gene
cluster region which is essential for the in vitro growth of M. tuberculosis. It is however not required for
the growth of the saprophytic mycobacterium M. smegmatis. In this study we have identified proteinprotein
interactions within the ESX-3 secretion system, using the Mycobacterial – Protein Fragment
Complementation (M-PFC) mycobacterial two-hybrid system, and created a model of the M.
tuberculosis ESX-3 secretion system. According to this model, the EsxG-EsxH and PE5-PPE4
substrate protein complexes bind to the same components of the ESX-3 secretion machinery and are
secreted via the same mechanism. A knock-out of the ESX-3 secretion system in M. smegmatis was
generated by homologous recombination to allow further research into the functions and properties of
this secretion system. This knock-out was used, together with wild-type M. smegmatis, to investigate
the secretion of the M. tuberculosis EsxH protein by the M. smegmatis ESX-3 secretion system.
The ESX-3 secretion system interactome may serve as a model for the ESX secretion systems and
assist in our understanding of this secretion machinery which is key to the virulence and survival of M.
tuberculosis and other pathogenic mycobacteria. Improved understanding of these mechanisms and
their role in pathogenicity and survival may provide means of interfering with the secretion machinery,
potentially leading to developments in the prevention and treatment of tuberculosis disease. / AFRIKAANSE OPSOMMING: Tuberkulose, wat veroorsaak word deur Mycobacterium tuberculosis, eis jaarliks ongeveer 2 miljoen
lewens. Ten spyte van uitgebreide navorsing oor tuberkulose en M. tuberculosis is min bekend oor die
meganismes van patogenisiteit van díe organisme. Die genoom van M. tuberculosis bevat vyf ESAT-6
geen groep gebiede wat elk proteïene kodeer wat ‘n toegewyde sekresie sisteem vorm. Ingesluit in elk
van díe geen groep gebiede is gene wat T-sel antigene, serien proteases, ATP-bindingsproteïene en
ander membraan-geassosieërde proteïene kodeer. Alhoewel dit bekend is dat sekere van hierdie
sekresie sisteme betrokke is by virulensie en fagosoom-ontsnapping, en dat delesie daarvan die
organisme attenueer, is die struktuur, substrate en funksies van die sisteme grootliks onbekend.
Kennis van die struktuur van die ESX sekresie sisteme sal ons verstaan van die meganismes van
mikobakteriele patogenisiteit verbeter en leidrade verskaf na maniere om in te meng by díe
meganismes van virulensie.
Die ESAT-6 geen groep gebied 3, wat die ESX-3 sekresie sisteem kodeer, is die enigste ESAT-6
geen groep gebied wat noodsaaklik is vir die in vitro groei van M. tuberculosis. Dit is egter nie nodig vir
die groei van die saprofitiese mikobakterium M. smegmatis nie. In hierdie studie het ons proteïenproteïen
interaksies van die ESX-3 sekresie sisteem geïdentifiseer, deur middel van die
Mikobakteriële - Proteïen Fragment Komplementasie (M-PFC) mikobacteriële twee-hibriede stelsel.
Die interaksies is gebruik om ‘n model van die M. tuberculosis ESX-3 sekresie sisteem te skep.
Volgens díe model bind die EsxG-EsxH en PE5-PPE4 substraat proteïen komplekse aan dieselfde
komponente van die ESX-3 sekresie apparaat en word deur dieselfde meganisme uitgevoer. ‘n
uitklopmutant van die ESX-3 sekresie sisteem word deur homoloë rekombinasie in M. smegmatis
gegenereer om verdere ondersoeke na die funksies en eienskappe van hierdie sekresie sisteem in
staat te stel. Hierdie uitklopmutant is tesame met die wilde-tipe M. smegmatis gebruik om die sekresie
van die M. tuberculosis EsxH proteïen deur die M. smegmatis ESX-3 sekresie sisteem te ondersoek.
Die ESX-3 sekresie sisteem interaktoom kan dien as ‘n model vir die ESX sekresie sisteme om te help
om ons kennis van hierdie sekresie apparaat, wat belangrik is vir die virulensie en oorlewing van M.
tuberculosis en ander patogeniese mikobakterieë, te verbeter. Kennis van hierdie meganismes en hul
rol in patogenisiteit en oorlewing mag maniere verskaf om by díe sekresie sisteme in te meng, wat
moontlik kan lei tot ontwikkelings in die voorkoming en behandeling van tuberkulose.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4841 |
| Date | 03 1900 |
| Creators | Newton-Foot, Mae |
| Contributors | Van Pittius, Nicolaas Claudius Gey, Warren, Robin Mark, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : University of Stellenbsoch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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