Streptococcus pneumoniae is one of the major human pathogens and causes one to two million deaths every year as a result of meningitis, pneumoniae and sepsis. The pneumococcal cell wall consists of peptidoglycan and teichoic acids and plays an important role in virulence. The peptidoglycan maintains shape and osmotic stability of the cell. The synthesis of peptidoglycan is an important target for antibodies. The teichoic acids (TA) are differentiated in wall teichoic acids (WTA) and lipoteichoic acid (LTA). Both, LTA and WTA, consist of identical subunits, implying that their precursors originate from the same synthesis pathway. The pneumococcal TAs are decorated with choline, a compound that is very rare among bacteria. Choline is essential for the growth of S. pneumoniae. The choline residues bound to the TAs serve as anchors for the class of the choline binding proteins (CBPs), which includes important virulence factors such as LytA and PspA. The enzymes, which are responsible for the choline metabolism, are encoded in the lie region. In the last three decades numerous basic tools for cell biology research have been established for model organisms as E. coli. Many of these tools were not available for S. pneumoniae. In this work, novel tools for cell biology and cell wall analysis were established. The new cell biology methods include time-lapse microscopy of live cells and cellular localisation of GFP-tagged proteins, as well as a new Zn-r-Inducible promoter for efficient gene depletion. Using these tools, the gene products of the lie locus have been localised, providing the first evidence that the loading of WTA with phosphorylcholine residues is a membrane-associated process catalysed by the LicDl and LicD2 enzymes. The new cell wall methods include a protocol for the isolation of WTA chains and determination of the muropeptide pattern from isolated peptidoglycan. In this work, WTA chains were isolated for the first time and analysed by mass spectrometry. Additionally, the muropeptide profile of the laboratory strain R6 was established and new modifications of the pneumococcal peptidoglycan were found, which have not been described before. These novel tools will be very valuable to study the cell biology and the cell wall of the human pathogen S. pneumoniae.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606730 |
| Date | January 2010 |
| Creators | Eberhardt, Alice |
| Publisher | University of Newcastle Upon Tyne |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0131 seconds