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 understood. 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 Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting 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 virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

Streptococcus pneumoniae is the major cause of community-acquired bacterial pneumonia, and it is also responsible for otitis media and meningitis (2). Capsular polysaccharides were the first virulence factors to be identified. The capsule is thought to protect the bacteria from the host immune system by preventing phagocytosis (17). Purified capsular extracts do not have an inflammatory or toxic effect (31, 32). Among the proteins considered to be virulence factors (17, 45) are pneumolysin (3, 7, 12), autolysin (4, 12, 56), hyaluronidase (5), pneumococcal surface protein A (PspA) (8), PsaA (6), neuraminidase (10), immunoglobulin A1 (IgA1) protease (46, 59), and pyruvate oxidase (55), although for some of them a role in virulence has not been demonstrated.Recent advances in the field of bacterial pathogenesis have allowed a large-scale identification of new virulence genes in different bacterial species. The methods developed are based on the concept that specific gene products are required for each stage of an infection process and that their expression is often regulated by the different environmental conditions encountered in the host. Mahan et al. (38) developed a system called IVET (in vivo expression technology) aimed at identifying bacterial genes that were preferentially expressed in the host during infection and were poorly transcribed under laboratory conditions. IVET was originally developed for use with Salmonella typhimurium (38) and then applied to Vibrio cholerae (11) and Pseudomonas aeruginosa (58). Hensel et al. (28) expanded the concept of tagging originally developed by Walsh and Cepko (57) to monitor the fate of clonally related neocortical cells during brain development, developing a strategy to identify virulence genes by negative selection. This system, called STM (signature-tagged mutagenesis), exploits a pool of transposons in which each transposon is tagged with a unique DNA sequence so that the resulting insertion mutants are marked with a different DNA sequence. This permits the identification of bacteria recovered from hosts infected with a mixed population of mutants.Tagged insertion mutants are combined into pools, which are used to infect the animals. At a defined time point, bacteria are recovered from the animals. Tag sequences are amplified from each pool by using a radioactive label before and after the infection. These two labeled tag probes are hybridized to filters containing spotted genomic DNA from all mutants of the corresponding pool. Mutants whose tags are positive for hybridization with the probe from the original pool and negative with the one from the recovered bacteria are considered to be virulence attenuated. This system was originally used to identify genes involved in virulence in S. typhimurium (28) and recently applied to Staphylococcus aureus and V. cholerae (15, 42).S. pneumoniae has been studied for many years, yet its virulence mechanisms are not fully understood (17). Therefore, we have modified the original STM methodology to discover novel virulence factors in S. pneumoniae. Initially, we attempted to use for this purpose the encapsulated type 3 strain GP119, obtained by transformation from the nonencapsulated avirulent laboratory strain Rx1. Surprisingly, when GP119 was tested for virulence in a mouse septicemia model, it was found to be not virulent (unpublished observations); this result indicates that in factors in addition to the capsule are required for virulence. We then selected the encapsulated type 19F strain G54, a recent clinical isolate. This strain was chosen because of its high transformation rate (47) induced by using the 17-residue competence-stimulating peptide (26) and its virulence in a mouse pneumonia model, as assessed in our laboratory. The following two major changes were introduced in the original STM protocol (28).(i) A library of tagged mutants was constructed by insertion-duplication mutagenesis using short random genomic DNA fragments inserted in a suicide plasmid vector bearing the molecular tag. The original transposon mutagenesis technique (28) was not applied to S. pneumoniae, since we did not expect transposons to insert randomly as shown for Streptococcus mutans (24) and Lactococcus lactis (49).(ii) While in the original method (28) the filters corresponding to each pool had spots of genomic DNA from each mutant obtained by transferring bacterial colonies to the filters (colony hybridization), we used filters containing amplified tags from each mutant. This modification was necessary since in our hands colony hybridization generated a high background giving rise to false positives.Using the modified STM technique, we identified 126 putative virulence genes from S. pneumoniae. Some of them corresponded to previously described pneumococcal virulence factors, while others showed homology to virulence genes found in other bacteria. In addition, we have identified genes not previously known to be involved in virulence.