Am. J. Respir. Cell Mol. Biol., Volume 22, Number 6, June, 2000 722-731

Pneumocystis carinii Uses a Functional Cdc13 B-Type Cyclin Complex during Its Life Cycle

Theodore J. Kottom, Charles F. Thomas Jr., Kamal K. Mubarak, Edward B. Leof, and Andrew H. Limper

Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care, and Internal Medicine, and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota

Pneumocystis carinii causes severe pneumonia in immunocompromised patients. Recent studies indicate that P. carinii uses a Cdc2 cyclin-dependent kinase to control its proliferation. To further study the regulation of the life cycle of P. carinii, we characterized the P. carinii B-type cyclin termed Cdc13, whose binding to Cdc2 is necessary for kinase activity. Antibodies to B-type cyclins (Cdc13) specifically immunoprecipitated Cdc2/ Cdc13 complexes with associated kinase activity from P. carinii extracts. To clone P. carinii cdc13, degenerate polymerase chain reaction was undertaken using primers generated from amino-acid motifs conserved in fungal Cdc13 proteins. This amplicon was used to obtain full-length genomic and complementary DNA (cDNA) clones. A specific synthetic peptide antibody generated to P. carinii Cdc13 further demonstrated differential Cdc2/Cdc13 activity over the life cycle of P. carinii, with greater activity in cysts compared with trophic forms of the organism. Finally, P. carinii cdc13 cDNA was used to rescue mutant Schizosaccharomyces pombe strains containing temperature-sensitive deficiencies of endogenous Cdc13 activity, thus verifying function of the P. carinii Cdc13 protein. Therefore, P. carinii contains a Cdc13 cyclin, which is variably active over its life cycle and which promotes fungal proliferation.

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