Pneumocystis carinii remains an important cause of pneumonia in immunocompromised patients including those with AIDS. Our previous studies indicate that attachment of P. carinii to respiratory epithelial cells is a prominent component of the organism's life cycle, which promotes growth of P. carinii while concurrently inhibiting proliferation, and hence repair, of lung epithelium. Our data further indicate that the life cycle of P. carinii is regulated by a cyclin dependent kinase control system. P. carinii contains a Cdc2 cyclin dependent kinase with potent activity in promoting fungal proliferation, as well as an associated Cdc13 cyclin and a regulatory Cdc25 phosphatase. Coordinated expression and activation of these molecules is necessary for eukaryotic cell cycle progression. Attachment of P. carinii to alveolar epithelium serves as an important trigger strongly promoting completion of the organism cell cycle through alteration of this regulatory machinery. Concurrently, we have shown that binding of P. carinii to epithelial cells inhibits lung cell Cdc2 activity and impairs epithelial cell proliferation. In the current proposal, we hypothesize that binding of P. carinii to epithelial cells modulates activity of cyclin-dependent dent kinases and their associated molecules, both in the organism and in lung cells. This concurrently promotes growth of P. carinii, while impairing proliferation of epithelial cells. This hypothesis will be investigated through five Specific Aims. First, we will evaluate mechanisms by which P. carinii binding to lung epithelial cells suppresses host cell cyclin dependent kinase activity and growth through altered interactions of host Cdc2 with its required cyclins, or through impaired function of Cdc25, which activates Cdc2. We will further determine the relative contributions of host adhesive proteins and will genetically identify P. carinii surface molecules which support attachment of the organism and which induce suppression of the epithelial cell cycle. Next, we will determine the activity of P. carinii's cyclin dependent kinase system (Cdc2, Cdc13, Cdc25) over the life cycle of the organism, and will investigate the effects of P. carinii-epithelial binding on activating these cell cycle control molecules in P. carinii. Finally, we will evaluate whether inhibiting the P. carinii cyclin dependent kinase system results in suppression of organism proliferation, representing a novel approach to control this infection. Better understanding of how P. carinii attachment to lung epithelial cells mediates alterations in the cell cycles of both the organism and the host promises to yield important new therapeutic insights into P. carinii pneumonia.
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