Large-scale identification of virulence genes from Streptococcus pneumoniae

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely und erstood. Recent technical advances in the field of bacterial pathogenesis ( in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salm onella genes involved in pathogenicity. A library of pneumococcal chromosom al fragments (400 to 600 bp) was constructed in a suicide plasmid vector ca rrying unique DNA sequence tags and a chloramphenicol resistance marker. Th e recent clinical isolate G54 was transformed with this library, Chloramphe nicol-resistant mutants were obtained by homologous recombination, resultin g in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virul ence-attenuated mutants. The regions flanking the chloramphenicol gene of t he attenuated mutants were amplified by inverse PCR and sequenced. The sequ ence analysis showed that the 200 mutants had insertions in 126 different g enes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding pro teases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequen ces that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were ind ividually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the exis tence of virulence factors specific for pneumonia.