A functional genomic analysis of type 3 Streptococcus pneumoniae virulence

Abstract

SummaryStreptococcus 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 analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signaturetagged mutants was created by insertion±duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eightysix mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.

Versatility of pneumococcal surface proteins

“…In a systemic and experimental mouse meningitis model of infection, the pneumococcal pavA knockout of strain D39 is substantially attenuated (Holmes et al, 2001;Pracht et al, 2005). The impact of PavA on pathogenesis has been independently indicated by STM experiments using a mouse pneumonia model of infection (Lau et al, 2001). In addition, PavA is most likely involved indirectly and in a fibronectin-independent manner in pneumococcal adherence to host cells.…”

Neutrophil Elastase Mediates Innate Host Protection against Pseudomonas aeruginosa

“…In vitro competition studies were performed following modifications of a previously described method (37). First, WT P. aeruginosa and its isogenic mutant OprF Ϫ were freshly grown to mid-log phase and adjusted to the same OD.…”

Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection

“…Modifications of the method include the use of pre-selected tagged transposons to construct the mutant library, which simplifies the screening significantly (Mei et al, 1997). STM screenings of a mutant library in more than one infection model have been performed (Coulter et al, 1998;Tsolis et al, 1999;Bispham et al, 2001;Lau et al, 2001). Such screenings provide valuable knowledge of whether the same virulence factors are involved in different types of infections.…”