The evolution of the Mycobacterium tuberculosis proteome in response to the development of drug resistance

Fortuin, Suereta (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79814
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