MOLECULAR BIOLOGY OF P CARINII GP120

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Pneumonia caused by the opportunistic pathogen Pneumocystis carinii is a primary cause of mortality in patients with acquired immunodeficiency syndrome and in other immunocompromised hosts. The long range goal of the proposed study is to identify antigens of P. carinii that are involved in acquired resistance to infection. Patients or infected animals that have recovered from P. carinii pneumonia (PCP) demonstrate an immune response to an abundant, immunodominant surface glycoprotein of P. carinii, which in the ferret model of PCP has an Mr of 120 kDa (gpl2O). This and other data indicate that the surface glycoprotein is important in the host-P. carinii interaction. The immediate focus of this proposal is to perform a molecular biological analysis of ferret P. carinii gp 120. There are three main aspects to the proposed study. Several recombinant cDNA clones, each encoding a portion of the cDNA for ferret P. carinii gpl2O, have been isolated using specific antibodies against native ferret P. carinii gpl2O. Nucleotide sequence analysis suggested that the cDNA clone's correspond to transcripts encoding at least two different isoforms of gpl2O. These results support the hypothesis that genetic polymorphism of ferret P. carinii gpl2O results in variation of the protein core of the molecule. To test this hypothesis, the full-length cDNAs corresponding to the predicted ferret P. carinii gpl2O isoforms will be cloned and sequenced. Analysis of the nucleotide sequences will establish the inter-relationship of the cDNA clones. Recent analyses of the karyotypes of rat and human P. carinii established that genetic variation exists in the P. carinii genome at the chromosomal level. To investigate the potential for heterogeneity in the chromosomal organization of the gp120 gene, the electrophoretic karyotype of ferret P. carinii will be determined by pulsed-field gel electrophoresis and the gp l2O-specific chromosomes will be mapped by Southern hybridization. After expression in eukaryotic and prokaryotic expression systems, the antigenic relatedness of glycosylated and non-glycosylated recombinant gpl2O to native gpl2O will be compared using immunological methods. Molecular characterization of ferret P. carinii gp 120 will be essential to understand the basis of its interaction with the host immune system. The data obtained from the proposed studies will contribute to the design of diagnostic reagents and immunotherapy for PCP.