CONTROL OF THE PNEUMOCYSTIS CARINII CELL CYCLE

卡氏肺孢子虫仍然是免疫功能低下患者包括艾滋病患者肺炎的重要原因。我们以前的研究表明，卡氏肺孢子虫与呼吸道上皮细胞的附着是生物体生命周期的重要组成部分，它促进卡氏肺孢子虫的生长，同时抑制肺上皮细胞的增殖，从而抑制肺上皮细胞的修复。我们的数据进一步表明，卡氏肺孢子虫的生命周期受周期蛋白依赖的激酶控制系统的调控。卡氏假单胞菌含有一个具有促进真菌增殖活性的CDc2细胞周期蛋白依赖的激酶，以及一个相关的CDc13细胞周期蛋白和一个调控的CDC25磷酸酶。这些分子的协同表达和激活是真核细胞周期进程所必需的。卡氏肺孢子虫附着在肺泡上皮细胞上是一个重要的触发因素，通过改变这一调节机制，有力地促进了机体细胞周期的完成。同时，我们发现卡氏肺孢子虫与上皮细胞的结合抑制了肺细胞的CDC2活性，并损害了上皮细胞的增殖。在目前的建议中，我们假设卡氏肺孢子虫与上皮细胞的结合调节了生物体和肺细胞中细胞周期蛋白依赖性的凹陷蛋白激酶及其相关分子的活性。这同时促进了卡氏肺孢子虫的生长，同时损害了上皮细胞的增殖。这一假设将通过五个具体目标进行调查。首先，我们将评估卡氏肺孢子虫与肺上皮细胞结合，通过改变宿主细胞CDc2与其所需的细胞周期蛋白的相互作用，或通过CDC25功能受损(激活CDc2)，抑制宿主细胞周期蛋白依赖性激酶活性和生长的机制。我们将进一步确定宿主黏附蛋白的相对贡献，并从基因上鉴定卡氏肺孢子虫表面分子，这些分子支持生物体的附着，并诱导抑制上皮细胞周期。接下来，我们将测定卡氏肺孢子虫细胞周期蛋白依赖的激酶系统(CDC2、CDC13、CDC25)在生物体整个生命周期中的活性，并将研究卡氏肺孢子虫与上皮细胞的结合对激活这些细胞周期控制分子的影响。最后，我们将评估抑制卡氏肺孢子虫细胞周期蛋白依赖的激酶系统是否会抑制生物体的增殖，这代表了一种控制这种感染的新方法。更好地了解卡氏肺孢子虫附着于肺上皮细胞如何调节机体和宿主细胞周期的变化，有望为卡氏肺孢子虫肺炎提供重要的新的治疗见解。

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.