The bacterial pathogen Chlamydia trachomatis causes Trachoma, the worlds leading cause of preventable blindness and is also responsible for the most common curable sexually transmitted disease in the UK and United States. C. trachomatis is an obligate intracellular organism characterised by a unique and complex growth cycle. Its study presents many challenges since it has historically been recalcitrant to genetic manipulation and growth in the absence of a host cell. Nevertheless, the sequencing of the C. trachomatis genome and its relatively small size by comparison to genomes from other bacterial pathogens, has paved the way for studies at the proteomic level. This thesis describes the development and application of proteomic approaches to study C. trachomatis L2. To survey the expressed chlamydial proteome, a combination of the qualitative approaches, 2-DGE, MudPIT and GeLC-MS/MS; and the quantitative approaches AQUA, iTRAQ and LC-MSE were used. Collectively, the approaches efficiently identified 648 expressed proteins, representing ~72% of the predicted proteome of C. trachomatis L2, from both the infectious (elementary body, EB) and replicating (reticulate body, RB) form of the pathogen. In the infectious EB, the entire set of predicted glycolytic enzymes were detected, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. Further, proteomic analysis of the RB form also uncovered biosynthetic enzymes for chlamydial cell wall synthesis, indicating that peptidoglycan is produced in some form during growth in host cells. Comparison of the quantitative approaches iTRAQ and LC-MSE demonstrated that LC-MSE quantitative data was significantly more robust and extensive relative to iTRAQ data. In addition to information on relative amounts of these proteins between the two forms, LC-MSE data also yielded the cellular concentration (molecules per cell) for 489 proteins. This extensive set of absolute quantitation data permits estimates of the energy invested in the synthesis of various classes of proteins. The results indicate that C. trachomatis devotes most of its energy into maintenance of the translational machinery. However, it also expends significant amounts of energy into making cell envelope components and a set of hitherto hypothetical proteins. These proteins, which account for the bulk of the energy invested by the intracellular RB form of the pathogen as it converts to the extracellular EB form, highlight the importance of absolute quantitation data for understanding the biological processing status of the cell. The datasets also revealed a large number of proteins that were differentially expressed between replicating RBs and infectious EBs, ranging from 8.4-fold down-regulation to 3.5-fold up-regulation. Consistent with transcriptomic studies (Belland et al., 2003), proteins involved in protein synthesis, ATP generation, central metabolism, secretion and nutrient uptake were predominant in the metabolically active RB at 15 h PI. Although many of the proteins in these functional categories were down-regulated in EBs, proteins required for glycolysis, central metabolism, protein synthesis, and type III secretion were present in significant amounts in EBs suggesting that the infectious EB is primed ‘ready-to-go’ upon contact with the host cell.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:577248 |
| Date | January 2012 |
| Creators | Skipp, Paul |
| Contributors | O'Connor, C. David |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/344259/ |
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