Browsing by Author "Fortuin, Suereta"
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- ItemThe evolution of the Mycobacterium tuberculosis proteome in response to the development of drug resistance(Stellenbosch : Stellenbosch University, 2013-03) Fortuin, Suereta; 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.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.
- ItemOn the impact of the pangenome and annotation discrepancies while building protein sequence databases for bacteria proteogenomics(Frontiers Media, 2019) Machado, Karla C. T.; Fortuin, Suereta; Tomazella, Gisele Guicardi; Fonseca, Andre F.; Warren, Robin Mark; Wiker, Harald G.; De Souza, Sandro Jose; De Souza, Gustavo AntonioENGLISH ABSTRACT: In proteomics, peptide information within mass spectrometry (MS) data from a specific organism sample is routinely matched against a protein sequence database that best represent such organism. However, if the species/strain in the sample is unknown or genetically poorly characterized, it becomes challenging to determine a database which can represent such sample. Building customized protein sequence databases merging multiple strains for a given species has become a strategy to overcome such restrictions. However, as more genetic information is publicly available and interesting genetic features such as the existence of pan- and core genes within a species are revealed, we questioned how efficient such merging strategies are to report relevant information. To test this assumption, we constructed databases containing conserved and unique sequences for 10 different species. Features that are relevant for probabilistic-based protein identification by proteomics were then monitored. As expected, increase in database complexity correlates with pangenomic complexity. However, Mycobacterium tuberculosis and Bordetella pertussis generated very complex databases even having low pangenomic complexity. We further tested database performance by using MS data from eight clinical strains from M. tuberculosis, and from two published datasets from Staphylococcus aureus. We show that by using an approach where database size is controlled by removing repeated identical tryptic sequences across strains/species, computational time can be reduced drastically as database complexity increases.
- ItemPhosphoproteomics analysis of a clinical mycobacterium tuberculosis Beijing isolate : expanding the mycobacterial phosphoproteome catalog(Frontiers Media, 2015) Fortuin, Suereta; Tomazella, Gisele G.; Nagaraj, Nagarjuna; Sampson, Samantha L.; Gey Van Pittius, Nicolaas C.; Soares, Nelson C.; Wiker, Harald G.; De Souza, Gustavo A.; Warren, Robin M.Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.