Characterising virulence factors from pathogenic bacteria using fluorescent reporters

Protein translocation systems are invaluable to pathogenic bacteria, facilitating the display of virulence factors on their surface or their release into the extracellular environment. Some protein export systems are ubiquitous and essential to cell survival whereas others are horizontally acquired on prophages or pathogenicity islands (PAI), in many cases providing the bacterium with pathogenic advantages. For the majority of the known protein export systems, their structure, function and secreted substrates have been characterised, yet some proteins have been identified that are secreted via unknown mechanisms. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of human foodborne disease worldwide. The pathogenesis of this bacterium is mainly attributed to the secretion of toxins and the presence of a Type III Secretion System (T3SS). The T3SS can translocate bacterial proteins, known as effectors, into the host cell which mediate an effect culminating in the formation of a characteristic attaching and effacing (A/E) lesion. This system is encoded on a horizontally acquired PAI termed the locus of enterocyte effacement (LEE). The LEE not only encodes the T3SS apparatus but also several effectors secreted by the system and transcription factors which regulate its expression. However, it was recently found that T3SS not only secretes LEE encoded effectors but can also secrete proteins encoded on other prophages present in the EHEC genome. Characterisation of these non-LEE encoded effectors is ongoing and this study investigates the expression, regulation and function of non-LEE encoded effector H1 (NleH1) and H2 (NleH2). NleH1 and NleH2 are secreted by the T3SS but are encoded on different prophages. This study demonstrates that expression of NleH1 and NleH2 is induced in the same in vitro conditions which stimulate the expression of the LEE but is diminished upon initial host cell contact in vitro. Transcription of nleH1 and nleH2 is dependant upon factors specific to E. coli O157:H7 and these factors are regulated by LEE encoded regulators Ler and GrlA, as they have a positive effect on nleH transcription. NleH1 and H2 are predicted serine/threonine protein kinases and are able to autophosphorylate. Yeast two hybrid screening and 2D differential gel electrophoresis did not elucidate a eukaryotic protein binding partner of NleH1 or NleH2. Transfection assays show that they do not have a significant effect upon NF-κB activation in vitro. Determining the expression, regulation and function of non-LEE encoded effectors contributes towards further understanding of how this pathogen causes disease. Streptococcus pneumoniae, also known as the pneumococcus, is another globally important human pathogen. It is a very diverse pathogen, with over 90 capsular serotypes and is naturally competent for DNA uptake. Pneumococcal pathogenesis is facilitated by the production of a pore-forming toxin, pneumolysin. Pneumolysin’s activities in pneumococcal pathogenesis extend beyond its cytolytic function as it can also activate the complement pathway and modulate the host cytoskeleton. Pneumolysin is a member of a conserved family of toxins known as the cholesterol dependant cytolysins but differs due to the lack of a secretion signal peptide within its sequence. This indicates that it is not secreted from the bacterium however it has been reported that some strains can release pneumolysin in a cell lysis-independent manner. Additional to this, pneumolysin can also localise to the cell wall, and this localisation is not strain dependent. This study characterised codon-optimised N-terminally labelled pneumolysin constructs and applied them to assess the localisation of pneumolysin. In addition, the importance of autolysin and genes which are co-transcribed with Ply upon the localisation/secretion of pneumolysin was investigated by construction of a pneumococcal strain carrying an autolysin-pneumolysin fusion which naturally occurs in equine strains. These genes were not required for the translocation of pneumolysin or its association with the cell wall. Growth of this strain, and its isogenic parent, in vitro at a low density and low temperature resulted in the pneumolysin being detected in the broth culture. This indicates that pneumolysin can be released from the cell wall and that this action is not dependant upon the genes which were deleted in the mutant. The distribution of pneumolysin on the pneumococcal surface was assessed with immunofluorescence, and LumioTM substrate fluorescence, microscopy and found to have a general distribution. As a contribution to future pneumococcal research, codon-optimised fluorescent protein reagents were developed and can be used as reporters for gene expression and protein localisation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo