"Streptococcus pneumoniae is a major pathogen causing invasive (pneumonia, meningitis, bacteraemia) and non-invasive (acute otitis media, sinusitis) disease in young children and in elderly and/or immuno-compromised adults. The molecular mechanisms underlying S. pneumoniae virulence are not fully understood. Single-cell methodologies, including the sub-cellular localization of proteins by fluorescence microscopy, have been instrumental in studying fundamental processes in bacteria and these methodologies are potentially of great use to investigate S. pneumoniae virulence. Recently, we have developed a cell biology toolkit for S. pneumoniae, allowing us to perform gene expression and protein localization studies at the single cell level in live cells. A major S. pneumoniae virulence factor is pneumolysin which is encoded by the ply gene. Pneumolysin is highly immunogenic and binds to cholesterol in the membrane of host cells and its haemolytic activity induces cell lysis. Pneumolysin is one of the best characterized virulence factors of S. pneumoniae, but surprisingly little is known about its transcriptional regulation. Interestingly, pneumolysin is produced in the cytoplasm of S. pneumoniae and can only be released by lysis of the producer. This represents a fascinating paradox: if all cells lyse at the same time, the clonal lineage becomes extinct. Thus, mechanisms must exist to ensure that not all cells release or produce pneumolysin at the same time. This proposal is aimed at unraveling the molecular mechanisms underlying heterogeneous pneumolysin gene regulation and release using single cell analytical techniques. The host institute has excellent knowledge and facilities to perform S. pneumococcus molecular genetics and cell biology, and together with the single cell biology expertise of the researcher, this partnership should provide important insights into S. pneumoniae pathogenesis."