MOLECULAR BIOLOGY OF PNEUMOCYSTIS CARINII

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Pneumocystis carinii is the major pulmonary pathogen in patients with the acquired immunodeficiency sysndrome (AIDS). Studies on the basic biology of the organism have been hampered by lack of an adequate in vitro cultivation system. In order to develop new approaches to the study of this organism, a recombinant DNA library has been prepared from rat Pneumocystis which enables the isolation of Pneumocystis nucleic acid sequences via nucleic acid and antibody screening. This library will be utilized to address the following aspects of Pneumocystis biology: 1. The taxonomic position of Pneumocystis carinii has long been a subject of debate. Most investigators have concluded that it is a protozoan, however several have suggested that it is a fungus. In this laboratory, by phylogenetic analysis of rat pneumocystis small-subunit rRNA sequence, we have shown that pneumocystis is a fungus closely related to the yeasts (see below). We plan to extend and refine this analysis by isolation and sequencing of several rat and human Pneumocystis genes. By comparison of rRNA and antigen genes for several isolates of Pneumocystis, it will be determined whether or not strains or species of pneumocystis exist. The existence of strains or species of Pneumocystis may have important implications in the acquisition and therapy of the disease. 2. Specific interactions between host cells and Pneumocystis appear to be required for pathogenesis. This process must involve Pneumocystis proteins that recognize the surface of lung epithelial cells. Recent studies have shown that the host reacts to a limited repertoire of Pneumocystis proteins. Two major antigens have been recognized, both of which appear to be surface glycoproteins. These antigens will be cloned by screening of a Pneumocystis expression library with monoclonal antibodies of defined specificity. The structural and functional roles which these proteins play will be studied by expression in Saccharomyces cerevisae. Attachment to type I pneumocytes, presumably the first step in pathogenesis, will be investigated by testing the ability of S. cerevisae expressing pneumocystis antigens to attach to pulmonary alveolar cells in culture. The long term-goal this project is a better understanding of this important human pathogen. Taxonomic analysis is crucial to develop new strategies for the control and therapy of PCP. In addition, this information will be important for defining methods of axenic cultivation of Pneumocystis and epidemiologic studies of Pneumocystis infection. Analysis of the structure and function of Pneumocystis antigens will aid in the epidemiologic studies and begin to define those determinants on Pneumocystis which stimulate an immune response. A clear understanding of the immune response to Pneumocystis is necessary to begin to study what defect that AIDS patients and malnourished infants have in common.