Browsing by Author "Gey Van Pittius, Nicolaas C."

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    Phosphoproteomics 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.
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    Recombination in pe/ppe genes contributes to genetic variation in Mycobacterium tuberculosis lineages
    (BioMed Central, 2016-02-29) Phelan, Jody E.; Coll, Francesc; Bergval, Indra; Anthony, Richard M.; Warren, Rob; Sampson, Samantha L.; Gey Van Pittius, Nicolaas C.; Glynn, Judith R.; Crampin, Amelia C.; Alves, Adriana; Bessa, Theolis B.; Campino, Susana; Dheda, Keertan; Grandjean, Louis; Hasan, Rumina; Hasan, Zahra; Miranda, Anabela; Moore, David; Panaiotov, Stefan; Perdigao, Joao; Portugal, Isabel; Sheen, Patricia; De Oliveira Sousa, Erivelton; Streicher, Elizabeth M.; Van Helden, Paul D.; Viveiros, Miguel; Hibberd, Martin L.; Pain, Arnab; McNerney, Ruth; Clark, Taane G.
    Background: Approximately 10 % of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. Results: To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. Conclusions: This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development.