Streptococcus pneumoniae is a highly diverse and adaptable opportunistic pathogen that can infect and colonise different niches within the human host to cause a wide range of invasive disease (sepsis and meningitis) and noninvasive disease (pneumonia, otitis media and sinusitis). The molecular mechanisms that contribute to the different patterns of pneumococcal infection remain largely unknown. This thesis aims to determine the physiological and proteomic responses that allow the pneumococcus to survive and adapt to invasive and non-invasive sites. The comparative proteomic analyses of clinical S. pneumoniae isolates recovered from blood cultures (classified as invasive site isolates) and mucosal surfaces such as sputum, skin and ear swabs (classified as non-invasive site isolates) was initiated. The pneumococci were grown in vitro under standard conditions and the total cellular bacterial proteins extracted and analysed using both gel based and non-gel based proteomic approaches. Analysis of the pneumococcal isolates by two-dimensional polyacrylamide gel electrophoresis (2DGE) revealed that a high degree of heterogeneity existed between the pneumococcal isolates particularly among isolates in the invasive site isolates. Differential patterns of protein synthesis were observed that discriminated the pneumococcal isolates according to their sites of isolation. These were proposed to be associated with the bacterial adaptation to invasive and non-invasive sites of infection. Mass spectrometry was used to identify selected significant (ANOVA, p < 0.05) protein spots, which were further categorised into functional groups by Gene Ontology analysis. An extension of the 2DGE data using an integrated approach comprising bioinformatics, surfome analysis and a shotgun proteomic workflow provided a comprehensive qualitative and quantitative analyses of the pneumococcal intracellular and cell-surface proteomes. Proteins potentially involved in pneumococcal niche-specific adaptation and surface proteins with potential for further investigation and inclusion in the pipeline of vaccine candidates were identified. Quantitative regulation of proteins involved in energy metabolism, genetic competence, stress response, surface adhesion and virulence were considered important for pneumococcal adaptation to invasive and non-invasive sites. The anatomical sites colonised by the pneumococcus vary in their V availability for iron. The 2DGE method was also used on selected pneumococcal isolates from the two sites of infection to define the proteome variability linked to the effect of iron starvation that may contribute to the different disease outcomes associated with pneumococcal infections. The iron restricted condition was generated by cation depletion of the growth medium using Chelex-100. Quantitative differences in protein abundance were demonstrated that correlated with pneumococcal adaptation to iron restriction. The identification of selected significant spots by liquid chromatography-mass spectrometry and systems biology analysis of the identified proteins contributed to the elucidation of the molecular mechanisms underlying pneumococcal survival under iron limitation. The expression/repression of proteins functionally associated with metal ion binding, oxidative stress response, translation and virulence mainly constituted the pneumococcal adaptive responses to growth under conditions of limited iron availability. The data presented in this thesis extended our understanding of the molecular events underlying pneumococcal physiological adaptation and provide the basis of future work in this area.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:767339 |
| Date | January 2018 |
| Creators | Bittaye, Mustapha |
| Contributors | Forbes, Kenneth ; Cash, Philip |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=240152 |
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