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.
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