Large-Scale Identification of Virulence Genes from Streptococcus pneumoniae

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Infection and Immunity, December 1998, p. 5620-5629, Vol. 66, No. 12
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Large-Scale Identification of Virulence Genes from Streptococcus pneumoniae

Alessandra Polissi,¹ Andrea Pontiggia,¹ Georg Feger,² Mario Altieri,¹ Harald Mottl,¹ Livia Ferrari,¹ and Daniel Simon¹^,^*

Department of Microbiology, Medicine Research Centre, Glaxo Wellcome S.p.A., 37100 Verona, Italy,¹ and Geneva Biomedical Research Institute, Glaxo Wellcome, Geneva, Switzerland²

Received 6 May 1998/Returned for modification 22 June 1998/Accepted 18 August 1998

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitisin children. Apart from the capsule, the virulence factors ofthis pathogen are not completely understood. Recent technicaladvances in the field of bacterial pathogenesis (in vivo expressiontechnology and signature-tagged mutagenesis [STM]) have alloweda large-scale identification of virulence genes. We have adaptedto S. pneumoniae the STM technique, originally used for the discoveryof Salmonella genes involved in pathogenicity. A library of pneumococcalchromosomal fragments (400 to 600 bp) was constructed in a suicideplasmid vector carrying unique DNA sequence tags and a chloramphenicolresistance marker. The recent clinical isolate G54 was transformedwith this library. Chloramphenicol-resistant mutants were obtainedby homologous recombination, resulting in genes inactivated byinsertion of the suicide vector carrying a unique tag. In a mousepneumonia model, 1.250 candidate clones were screened; 200 ofthese were not recovered from the lungs were therefore consideredvirulence-attenuated mutants. The regions flanking the chloramphenicolgene of the attenuated mutants were amplified by inverse PCR andsequenced. The sequence analysis showed that the 200 mutants hadinsertions in 126 different genes that could be grouped in sixclasses: (i) known pneumococcal virulence genes; (ii) genes involvedin metabolic pathways; (iii) genes encoding proteases; (iv) genescoding for ATP binding cassette transporters; (v) genes encodingproteins involved in DNA recombination/repair; and (vi) DNA sequencesthat showed similarity to hypothetical genes with unknown function.To evaluate the virulence attenuation for each mutant, all 126clones were individually analyzed in a mouse septicemia model.Not all mutants selected in the pneumonia model were confirmedin septicemia, thus indicating the existence of virulence factorsspecific forpneumonia.

^* Corresponding author. Mailing address: Glaxo Wellcome S.p.A., Microbiology Department, via A. Fleming 4, 37135 Verona, Italy.Phone: (39) 45 9218554. Fax: (39) 45 9218196. E-mail: ds20886{at}glaxowellcome.co.uk.

Infection and Immunity, December 1998, p. 5620-5629, Vol. 66, No. 12
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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