Studies on pneumolysin and on pneumococcal gene expression under aerobiosis

In this thesis two aspects of Streptococcus pneumoniae were studied. First, studies were done on the pneumolysin toxin of the pneumococcus, which has been shown to play an important role in the development of disease. This toxin is known to possess cytolytic and complement activation activities, which contribute to virulence in different ways. However, mutational studies showed that pneumolysin also contributes to virulence by a still unknown function. Following in vitro studies with a truncated pneumolysin it was reported that independent of its cytolytic activity, pneumolysin induces the production of interferon-y and nitric oxide from spleen cells. It was hypothesised that this could be the third contribution to virulence of pneumolysin. In the first project, this hypothesis was tested by studying the behaviour and virulence, in mouse models of disease, of a pneumococcal mutant that expressed a truncated pneumolysin. The hypothesis was not confirmed and the production of interferon-y and nitric oxide in the infected mice was found independent of the presence of pneumolysin. Interestingly, it was also found that the pneumococcal mutant was less virulent than a mutant that completely lacked the production of pneumolysin. The pneumococcus is an aerotolerant anaerobe that usually infects through the nasopharynx. There it must survive in the oxygen rich environment to persist in the infected host. The second project of this thesis aimed to understand how the pneumococcus is capable of adapting to the presence of oxygen. This was done by the use of microarrays to compare the gene expression of aerobically and anaerobically grown bacteria. The results showed 55 genes up-regulated and 14 genes down-regulated when S. pneumoniae was subject to aerobiosis. Gene rgg, which was up-regulated during aerobiosis, has been found in other bacteria to be involved in biofilm formation, energy metabolism and oxidative and thermal stress. This gene was chosen for further study. A pneumococcal rgg knockout mutant was constructed and in vitro growth under aerobic and anaerobic conditions studied