A functional genomic analysis of type 3 Streptococcus pneumoniae virulence

Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature-tagged mutagenesis together with an analys is of S. pneumoniae genome sequence to identify and characterize genes requ ired for pathogenesis. A library of signature-tagged mutants was created by insertion-duplication mutagenesis, and 1786 strains were analysed for thei r inability to survive and replicate in murine models of pneumonia and bact eraemia. One hundred and eighty-six mutant strains were identified as atten uated, and 56 were selected for further genetic characterization based on t heir ability to excise the integrated plasmid spontaneously. The genomic DN A inserts of the plasmids were cloned in Escherichia coli and sequenced. Th ese sequences were subjected to database searches, including the S. pneumon iae genome sequence, which allowed us to examine the chromosomal regions fl anking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were iden tified. Five strains mutated in genes involved in gene regulation, cation t ransport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional exp eriments also suggest that induction of competence for genetic transformati on has a role in virulence.