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Type: Thesis
Title: The role of CpsC in the regulation of Streptococcus pneumoniae capsular polysaccharide biosynthesis.
Author: Byrne, James
Issue Date: 2012
School/Discipline: School of Molecular and Biomedical Science
Abstract: Streptococcus pneumoniae is the leading cause of pneumonia, bacteraemia (sepsis), meningitis and otitis media worldwide. It is also responsible for more deaths worldwide than any other single pathogen. A major component of pneumococcal virulence is determined by the composition of polysaccharide layer that covers the bacteria, its capsule. The composition of the pneumococcal capsule provides the basis for the classification of the pneumococcus and to date 91 structurally distinct capsular serotypes have been identified. The capsule is essential for the virulence of the pneumococcus and its primary role during pathogenesis is to act as a physical barrier preventing the interaction of cell wall bound opsonins and their cognate receptors on phagocytes, thereby preventing phagocytosis. However, during colonisation, the CPS masks adhesions suggesting the amount of CP expressed on the cell surface needs to be carefully regulated. The regulation of capsule biosynthesis is mediated by the actions of four gene products, CpsA, B, C and D. CpsB, C and D form a series of complex interactions involved in the control of capsule subunit export and chain length regulation while CpsA has recently implicated in the ligation of capsular polysaccharide to the cell wall. A transphosphorylative event at the C-¬≠terminus of CpsD (facilitated by the presence of CpsC) results in a conformational change promoting the ligation of exported capsule subunits to the cell wall via an unknown mechanism. CpsB the de-phoshorylates CpsD resulting in a conformational change conducive to capsule export and polymerization. CpsC has been shown to be essential for the activity of CpsD. The interactions CpsC forms with other proteins involved in capsule biosynthesis have not been fully characterized and this study sought to analyse CpsC function by performing site directed mutagenesis on cpsC and analysing phenotypic changes. Characterisation of these changes and further analysis was used to attempt to determine the structure and oligomeric nature of CpsC under native membrane conditions. Attempts to use computer modelling techniques to better resolve the structure and oligomeric state of CpsC were also made. We have shown that defined single amino acid substitutions in different regions of CpsC can alter the phenotype of D39 to either mucoid or small colony phenotypes. In particular we have highlighted several domains including a region preceding and within the second transmembrane region of Cps2C that, when mutated, results in significant reductions in capsule biosynthesis. When the constructed mutants were tested in an in vitro adherence and invasion model (A549 cell monolayers) no consistent correlation of capsule quantity to either adherence or invasion was observed. Further characterisation of bacterial surface protein availability in these mutants did not align with either capsule quantity or the adherence and invasion results. Structural analysis of CpsC using computer modelling and experimental approaches highlighted the sequence conservation between members of the PCP protein family but did not uncover the structural form of CpsC. However, solubilisation of CpsC in its native membrane arrangement and analysis using various proteomic methods suggests the formation of an oligomer comprised of 4-6 monomer subunits as is consistent in the existing literature.
Advisor: Paton, James Cleland
Morona, Renato
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2012
Keywords: bacteria; Streptococcus pneumoniae; pneumococcus; CpsC; polysaccharide; capsular polysaccharide; exopolysaccharide
Provenance: Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
Appears in Collections:Research Theses

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