Truncated forms of PspA that are secreted from Streptococcus pneumoniae and their use in functional studies and cloning of the pspA gene.
Insertion-duplication mutagenesis was used to generate mutants of Streptococcus pneumoniae that produced truncated forms of PspA (pneumococcal surface protein A). The truncated products, representing from 20 to 80% of the complete PspA molecule, were all secreted from the cell and could be detected in unconcentrated culture medium. Analysis of the truncated molecules showed that the antigenic variability known to be associated with PspA is located in the alpha-helical N-terminal half of the molecule. This region was also found to contain immunogenic and protection-eliciting epitopes and to define the maximum region of the molecule that is likely to be surface exposed. The apparent molecular weight variability seen for PspA molecules of different S. pneumoniae strains was localized to both the N- and C-terminal halves of the protein. Attachment of PspA to S. pneumoniae was found to require regions located carboxy to the fifth repeat unit in the C-terminal end of the molecule. From the insertion-duplication mutants, the complete pspA gene was cloned and expressed in Escherichia coli. Differences in apparent molecular weight were observed when the same cloned product was expressed in E. coli and S. pneumoniae, suggesting that PspA is modified differently in the two hosts.
Yother
J
J
Department of Microbiology, University of Alabama, Birmingham 35294.
Handsome
G L
GL
Briles
D E
DE
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D001426:Bacterial Proteins; C051592:pneumococcal surface protein A