Control of the Pneumocystis carinii Life Cycle

描述(由申请人提供)：肺孢子虫肺炎仍然是免疫功能低下患者死亡和发病的重要原因。我们的研究表明，肺孢子虫与肺泡上皮细胞的结合是感染的一个中心特征，导致生物增殖。肺孢子虫附着于上皮细胞是通过宿主细胞外基质蛋白，特别是纤维连接蛋白和玻璃体连接蛋白介导的。我们最近证实，肺孢子虫表达一个INT1基因，它与整合素类分子同源，促进生物体对基质涂层表面的黏附。我们的研究进一步表明，肺孢子虫与肺细胞或基质的结合特异性地诱导了生物体中特定的信号通路的表达，最明显的是STE20和CBK1。有趣的是，这两种酶都具有调节β-葡聚糖细胞壁组装和刺激真菌增殖的关键活性。因此，我们假设肺孢子虫与肺细胞外基质蛋白和上皮细胞结合，激活特定的激酶信号级联反应，刺激生命周期进程，促进生物体的细胞壁组装和增殖。这些概念将通过四个独立但相互关联的具体目标加以处理。在目标1中，我们将确定肺孢子虫INT-1在调节机体与肺上皮细胞黏附中的作用。在目标2中，我们将评估肺孢子虫黏附于基质和肺细胞促进上游肺孢子虫STE20激酶激活的机制，并将确定STE20与生物体的下游CBK(细胞壁生物合成激酶)的相互作用。接下来，目标3将评估肺孢子虫的黏附是否会促进生物体中β-葡聚糖细胞壁组装机制的表达和活性，从而促进营养形式向包囊的过渡，包囊是生物体生命周期的关键组成部分。最后，在目标4中，我们将利用肺孢子虫中活性的细胞壁组装抑制物，进一步剖析免疫抑制的啮齿动物接种纯化的营养肺孢子虫后的生活史进程和感染情况。我们将确定细胞壁组装抑制对这些动物中与(-葡聚糖)细胞壁生成相关的各自营养形式和包囊种群的影响。特异性地阻断肺孢子虫与肺上皮细胞的黏附和抑制包囊，减少生物的增殖，可能会产生预防和治疗肺孢子虫肺炎的新的治疗方法。

DESCRIPTION (provided by applicant): Pneumocystis pneumonia remains a significant cause of mortality and morbidity in immunocompromised patients. Our studies have demonstrated that binding of Pneumocystis to alveolar epithelial cells is a central feature of infection, leading to organism proliferation. Pneumocystis attachment to epithelial cells is mediated through host extracellular matrix proteins, particularly fibronectin and vitronectin. We recently demonstrated that Pneumocystis expresses an INT1 gene, homologous to integrin-like molecules, which promotes organism adherence to matrix-coated surfaces. Our investigations further reveal that binding of Pneumocystis to lung cells or matrix specifically induces expression of particular signaling kinases in the organism, most notably STE20 and CBK1. Interestingly, both of these kinases have critical activities in regulating beta-glucan cell wall assembly and stimulating proliferation of fungi. We, therefore, hypothesize that binding of Pneumocystis to lung extracellular matrix proteins and epithelial cells, activates specific kinase signaling cascades that stimulate life cycle progression, promoting cell wall assembly and proliferation of the organism. These concepts will be addressed through four independent but interrelated specific aims. In Aim 1, we will define the role of Pneumocystis INT-1 in mediating adherence of the organism to lung epithelial cells. Under Aim 2, we will assess the mechanisms by which Pneumocystis adherence to matrix and lung cells promotes activation of the upstream Pneumocystis STE20 kinase, and will determine the interactions of STE20 with the downstream CBK (Cell Wall Biosynthesis Kinase) of the organism. Next, Aim 3 will evaluate whether Pneumocystis adherence will promote expression and activity of the beta-glucan cell wall assembly machinery in the organism, with subsequent transition of trophic forms to cysts, a critical component of the organism's life cycle. Finally in Aim 4, we will utilize cell wall assembly inhibitors, which are active in Pneumocystis, to further dissect life cycle progression and infection in immune suppressed rodents inoculated with purified Pneumocystis trophic forms. We will determine the effect of cell wall assembly inhibition on respective trophic form and cyst populations associated with (-glucan cell wall generation in these animals. Specific interruption of Pneumocystis adherence to lung epithelial cells and suppression of encystment, with reduced organism proliferation, may yield novel therapeutic approaches for the prevention and management of Pneumocystis pneumonia.