Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study is the first of its kind to highlight the importance of using the latest state of
the art technology available in the field of proteomics as a complementary tool to
characterize the proteome of members of the Mycobacterium tuberculosis Beijing
lineage which have been linked to outbreaks and drug resistance of Tuberculosis
(TB).
Our label-free comparative analysis of two closely related M. tuberculosis strains with
different transmission patterns and levels of virulence highlighted numerous factors
that may alter metabolic pathways leading to hyper-virulence whereby the strain was
able to rapidly replicate in the host and cause extensive disease. This comparative
analysis clearly demonstrated that both instrumentation and analysis software impacts
on the number of proteins identified and thereby the interpretation of the proteomic
data. These proteomes also served as substrates for the discovery of phosphorylation
sites, a field of research that reflects a significant knowledge gap in the field of M.
tuberculosis. By using differential separation techniques in combination with the
state of the art mass spectrometry we described the phosphorylation sites on 286
proteins. This was the first study to document phosphorylation of tyrosine residues in
M. tuberculosis. By this means, our data set further extend and complement previous
knowledge regarding phosphorylated peptides and phosphorylation sites in M.
tuberculosis. Using advanced mass spectrometry methods we further investigated the impact of the
in vivo evolution of rifampicin resistance on the proteome of a rifampicin-resistant
strain containing a S531L rpoB mutation. We identified the presence of overabundant
proteins which could provide novel insight into potential compensatory mechanisms that the bacillus uses to reduce susceptibility to anti-TB drugs. Our
findings suggest that proteins involved in a stress response may relate to an altered
physiology enabling the pathogen to tolerate and persist when exposed to anti-TB
drugs. Together this suggests that structural changes in the RNA polymerase
precipitated a cascade of events leading to alterations of metabolic pathways. In
addition, we present the first comprehensive analysis of the effect of rifampicin on the
proteome of a rifampicin resistant M. tuberculosis isolate suggesting that rifampicin
continues to influence the biology of M. tuberculosis despite the presence of an rpoB
mutation. Our analysis showed alterations in the cell envelope composition and
allowing the bacterium to survive in a metabolically dormant/persistent growth state.
The results presented in this study illustrate the full potential of using a proteomic
approach as a complementary molecular technique to select promising candidate
molecules and genes for further characterization using the tools of molecular biology. / AFRIKAANSE OPSOMMING: Die huidige studie is ‘n eerste van sy soort, deur die nuutste gevorderde tegnologie in
die proteomika veld te gebruik. Die proteoom van lede van die Mycobacterium
tuberculosis Beijing stam, wat die oorsaak is van tuberkulose (TB) uitbrake en ook
weerstandige TB, is gekarakteriseer.
Ons merkervrye vergelykende analise van twee naby verwante M. tuberculosis
stamme met verskillende vlakke van oordraagbaarheid en virulensie, beklemtoon
verskeie faktore wat metaboliese paaie mag verander, wat kan ly tot hiper-virulensie,
wat die TB-stam in staat stel om vinniger te repliseer in die gasheer en ‘n uitgebreide
siektetoestand kan veroorsaak. Die analise het duidelik gewys dat die toerusting wat
gebruik word, sowel as die sagteware ‘n invloed kan hê op die hoeveelheid proteïne
wat geïdentifiseer kan word en daardeur intrepretasie van proteomika data kan
beïnvloed. Hierdie proteome dien as substrate vir die ondekking van fosforilasie
setels, ‘n veld van navorsing wat dui op ‘n gaping in ons kennis van M. tuberculosis.
Deur gebruik te maak van differensiële skeidingstegnieke en moderne spektrometrie
beskryf ons fosforileringsetels in 286 proteine. Hierdie is die eerste studie wat
fosforilasie van tirosien residue in M. tuberculosis beskryf. Hierdeur komplimenteer
en brei ons data die huidige kennis oor gefosforileerde peptiede en fosforilasie setels
in M. tuberculosis uit. Deur gebruik te maak van gevorderde massa spektrometriese tegnieke het ons verder
ook die impak van in vivo evolusie van rifampicin weerstandigheid op die proteoom
van ‘n rifampicin weerstandige TB-stam met die algemene S531L rpoB mutasie
ondersoek. Ons het proteïne geïdentifiseer wat in groot hoeveelhede voorkom en kan nuwe insigte gee tot potensiele kompenserende meganismes wat deur die bacillus
gebruik word om vatbaarheid vir anti-TB middels te verminder. Ons bevindings dui
daarop dat proteïene betrokke in ‘n stresreaksie mag lei tot ‘n verandering in fisologie
wat die patogeen in staat stel om anti-TB middels te verdra en te volhard in die
teenwoordigheid van sulke middels. Saam impliseer dit dat ‘n ketting van gebeure
wat lei tot veranderinge in metaboliese paaie, word vooraf gegaan deur strukturele
veranderinge in die RNS polimerase. Tesame hiermee bied ons ook die eerste
omvattende analise aan van die effek wat rifampicin op die proteoom van ‘n
rifampicin weerstandige M. tuberculosis isolaat het, en wat aan die hand doen dat
rifampicin voordurend die biologie van M. tuberculosis beïnvloed, ten spyte van die
teenwoordigheid van ‘n rpoB mutasie. Ons analise dui op veranderinge in die
samestelling van die selomhulsel wat die bakterie toelaat om te oorleef in ‘n
metabolies dormante staat.
Die resultate wat in hierdie studie aangebied word illustreer die volle potensiaal van
‘n proteomiese benadering as komplementêre molekulêre tegniek om belowende
kandidaat molekules en gene te kies vir verdere karakterisering, deur gebruik te maak
van molekulêre tegnieke. / The National Research Foundation (RSA), / Norwegian Research Council (Norway) / National Institute of Health –Forgarty (USA) / Southern Africa Consortium for Research Excellence-Welcome Trust (SACORE) (United Kingdom) / Kwazulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) (USA)
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79814 |
| Date | 03 1900 |
| Creators | Fortuin, Suereta |
| Contributors | Warren, Robin M., Gey van Pittius, Nicolaas C., Wiker, Haraald G., De Souza, Gustavo A., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xvii, 143 p. : ill. |
| Rights | Stellenbosch University |
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