Genetic analysis of virulence of Streptococcus pneumoniae

Streptococcus pneumoniae (the pneumococcus) is known to be a genetically diverse species, and this important pathogen is amongst the most significant causes of bacterial disease in humans (such as pneumonia, bacteraemia, meningitis and otitis media). The infection process exposes the pneumococcus to numerous stress conditions, including temperature shift between the upper respiratory tract and deeper tissues, pH changes, exposure to reactive oxygen generated by host phagocytes, and nutritional deprivation, hi order to survive, bacteria must have the ability to sense and respond to their environment in the host. A central role in this environment response is played by the two-component systems (TCS). The S. pneumoniae genome sequence contains 13 putative complete TCS and one orphan response regulator. The project described in this thesis is aimed at the investigation of the regulation of expression of pneumococcal genes by TCS06 and TCS09 including those which are important to the virulence of this pathogenic bacteria. Furthermore, the putative virulence factor dlt operon regulated or potentially regulated by CiaR/H system was studied to investigate its contribution to the role of CiaR/H virulence. In addition, comparative genomic hybridization (CGH) was performed to investigate the genetic diversity in a collection of clinical isolates including several capsule serotype 14 and evaluate the diversity in virulence genes of the bacteria. TCS06 was found to be important for the ability of the pneumococcus to invade the lungs and blood in a murine model of disease but it does not affect the overall outcome of pneumococcal disease. The phenotype associated with deficiency of rr06 shows that the TCS06 is important for the bacteria when grown at higher temperatures. The transcriptional profile of a pneumococcal mutant lacking the response regulator of TCS06 found by microarray analysis allowed us to determine which transcriptional changes were occurring. The TCS09 was found to be attenuated in TIGR4 strain after intranasal infection in a murine model of infection. Microarray comparison of the transcriptional profiles of the wild-type strains with the rr09 mutants showed that TCS09 appeared to (directly or indirectly) regulate different genes in D39 and TIGR4 strains. The dlt operon was found to be essential for bacterial growth at higher temperatures. Furthermore, this operon was shown to be involved in the acid tolerance response and sensitivity to antimicrobial peptides. However, no attenuation was found in murine model of disease using TIGR4 strain lacking the dltA gene. CGH of thirteen pneumococcal isolates showed that reference strain TIGR4 contains twenty-five regions of diversity not shared in at least one of the strains tested, and three of these regions were identified for the first time in this study. In this study we provide a clear demonstration of genetic differences between strains of the same capsule serotype and ST. Furthermore, we show that clonal strains with the same serotype and ST behave differently in an animal model