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

[摘要] ENGLISH ABSTRACT: This study is the first of its kind to highlight the importance of using the latest state ofthe art technology available in the field of proteomics as a complementary tool tocharacterize the proteome of members of the Mycobacterium tuberculosis Beijinglineage which have been linked to outbreaks and drug resistance of Tuberculosis(TB).Our label-free comparative analysis of two closely related M. tuberculosis strains withdifferent transmission patterns and levels of virulence highlighted numerous factorsthat may alter metabolic pathways leading to hyper-virulence whereby the strain wasable to rapidly replicate in the host and cause extensive disease. This comparativeanalysis clearly demonstrated that both instrumentation and analysis software impactson the number of proteins identified and thereby the interpretation of the proteomicdata. These proteomes also served as substrates for the discovery of phosphorylationsites, a field of research that reflects a significant knowledge gap in the field of M.tuberculosis. By using differential separation techniques in combination with thestate of the art mass spectrometry we described the phosphorylation sites on 286proteins. This was the first study to document phosphorylation of tyrosine residues inM. tuberculosis. By this means, our data set further extend and complement previousknowledge regarding phosphorylated peptides and phosphorylation sites in M.tuberculosis.Using advanced mass spectrometry methods we further investigated the impact of thein vivo evolution of rifampicin resistance on the proteome of a rifampicin-resistantstrain containing a S531L rpoB mutation. We identified the presence of overabundantproteins which could provide novel insight into potential compensatory mechanisms that the bacillus uses to reduce susceptibility to anti-TB drugs. Ourfindings suggest that proteins involved in a stress response may relate to an alteredphysiology enabling the pathogen to tolerate and persist when exposed to anti-TBdrugs. Together this suggests that structural changes in the RNA polymeraseprecipitated a cascade of events leading to alterations of metabolic pathways. Inaddition, we present the first comprehensive analysis of the effect of rifampicin on theproteome of a rifampicin resistant M. tuberculosis isolate suggesting that rifampicincontinues to influence the biology of M. tuberculosis despite the presence of an rpoBmutation. Our analysis showed alterations in the cell envelope composition andallowing the bacterium to survive in a metabolically dormant/persistent growth state.The results presented in this study illustrate the full potential of using a proteomicapproach as a complementary molecular technique to select promising candidatemolecules and genes for further characterization using the tools of molecular biology.

[发布机构] Stellenbosch University