Control of the Pneumocystis carinii Life Cycle

描述（由申请人提供）：肺囊虫性肺炎仍然是免疫功能低下患者死亡和发病的重要原因。我们的研究表明肺囊虫与肺泡上皮细胞的结合是感染的核心特征，导致生物体增殖。肺囊虫附着于上皮细胞是通过宿主细胞外基质蛋白介导的，特别是纤维连接蛋白和玻璃体连接蛋白。我们最近证明肺囊虫表达一个INT1基因，与整合素样分子同源，促进生物体粘附在基质表面。我们的研究进一步表明，肺囊虫与肺细胞或基质的结合特异性地诱导了生物体中特定信号激酶的表达，最显著的是STE20和CBK1。有趣的是，这两种激酶在调节β -葡聚糖细胞壁组装和刺激真菌增殖方面具有关键活性。因此，我们假设肺囊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，刺激生命周期的进展，促进细胞壁组装和生物体的增殖。这些概念将通过四个独立但相互关联的具体目标来解决。在目的1中，我们将定义肺囊虫INT-1在介导有机体粘附到肺上皮细胞中的作用。在Aim 2中，我们将评估肺囊虫附着于基质和肺细胞促进上游肺囊虫STE20激酶活化的机制，并将确定STE20与生物体下游CBK（细胞壁生物合成激酶）的相互作用。接下来，Aim 3将评估肺囊虫粘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，并随后将营养形式转变为囊肿，这是生物体生命周期的关键组成部分。最后，在Aim 4中，我们将利用在肺囊虫中有活性的细胞壁组装抑制剂，进一步解剖接种纯化肺囊虫营养形式的免疫抑制啮齿动物的生命周期进展和感染。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态和囊肿种群的影响。特异性阻断肺囊虫对肺上皮细胞的粘附和抑制包囊，减少机体增殖，可能为肺囊虫性肺炎的预防和管理提供新的治疗方法。

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.