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. 1998 Dec;66(12):5620-9.
doi: 10.1128/IAI.66.12.5620-5629.1998.

Large-scale identification of virulence genes from Streptococcus pneumoniae

A Polissi  1 , A Pontiggia, G Feger, M Altieri, H Mottl, L Ferrari, D Simon
Affiliations

Large-scale identification of virulence genes from Streptococcus pneumoniae

A Polissi et al. Infect Immun. 1998 Dec.

Abstract

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.

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Figures

FIG. 1
FIG. 1
(A) Construction of a pool of pR326 plasmid DNA tags. A pool of double-stranded DNA tags was generated by extending with T7 DNA polymerase the 18-mer primer P1 annealed to random 89-mer templates (see Materials and Methods). Each tag is a variable sequence of 40 bp (N40), where N is A, C, G, or T, flanked by two constant arms of 20 bp which contain restriction sites for HindIII and KpnI. These arms allow PCR amplification with the upper primer (U.P.) and lower primer (L.P.) of each tag. Tags were digested with KpnI and ligated in the vector pR326, generating a pool of tagged plasmids. (B) Construction of a library of S. pneumoniae-tagged mutants by homologous recombination. S. pneumoniae chromosomal DNA was mechanically sheared; fragments ranging from 500 to 700 bp were size selected and ligated with the pool of pR326 plasmids DNA tags. The ligation mix was directly transformed in S. pneumoniae G54; homologous recombination events lead to insertion-duplication mutagenesis, with the plasmid carrying the cat gene and the tag inserted into the inactivated gene in the chromosome. Total genomic DNA was extracted from mutant clones, digested with a restriction endonuclease, and circularized by ligation. The genomic flanking region outside the cat gene was amplified with the inner set of primers cat0 and cat770; 4 ng of the product of the first PCR was reamplified with the nested pair of primers cat1 and cat720. The resulting single-PCR product was then sequenced.
FIG. 2
FIG. 2
Southern blot analysis of mutants. Genomic DNAs obtained from clones 23, 187, 985, and 1177 were digested with EcoRV (E), or with EcoRV and NcoI (E/N). The EcoRV site is absent in pR326; the NcoI site is unique to the cat gene. Five micrograms of digested DNA was then applied on 0.8% agarose gel, electrophoresed, blotted on nylon filter, and subjected to Southern hybridization analysis using the cat gene as a probe. G54 genomic DNA did not hybridize to the probe (data not shown). The positions of size markers are shown in kilobases on the right.
FIG. 3
FIG. 3
Design of virulence gene screening. S. pneumoniae chromosomal DNA was fragmented, size selected, and ligated with the pool of pR326 plasmid DNA tags. Homologous recombination events lead to insertion-duplication mutagenesis. DNA tags were individually amplified from all mutants and applied to duplicate filters in groups of 50; 50 mutants representing the tags on each filter were pooled and grown in a flask (20 ml of broth). Fifteen milliliters was removed for DNA extraction (in vitro pool). Fifty microliters was used to inoculate mice intranasally; after 32 h, the mice were sacrificed and their lungs were removed and homogenized. The recovered bacteria (in vivo pool) were plated by serial dilutions. The bacteria recovered (approximately 50,000 CFU from each mouse) were pooled and grown in a flask, and then the genomic DNA was extracted. The tags of the in vitro and in vivo pools were amplified and labeled with [32P]dCTP. Variable regions were recovered by HindIII digestion and used to probe twin filters; DNA tags that hybridize to the family of probes from the in vitro pool but not from the in vivo pool correspond to mutants negatively selected in vivo.
FIG. 4
FIG. 4
Identification of avirulent mutants. Two identical filters containing the same amount of individually amplified DNA tags (from A1 to L5) were hybridized to labeled DNA tags from the in vitro pool and the in vivo pool. The filters shown exemplify the many analyzed. Tags that weakly hybridized to the probes from both the in vitro pool and the in vivo pool (D1, E1, F1, G1, D2, C5, and E3) probably corresponded to slow-growing mutants and were not considered. Tags that hybridized to the probe from the in vitro pool but not to the probe from the in vivo pool (I5, L5, F5, L1, A2, and C4) corresponded to mutants with attenuated virulence and therefore were unable to survive in the host.
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