Pneumocystis carinii Interactions with Lung Epithelial Cells and Matrix Proteins Induce Expression and Activity of the PcSte20 Kinase with Subsequent Phosphorylation of the Downstream Cell Wall Biosynthesis Kinase PcCbk1

摘要

Eukaryotic cell proliferation and phenotype are highly regulated by contact-dependent mechanisms. We have previously shown that the binding and interaction of the opportunistic fungal pathogen Pneumocystis carinii to lung epithelial cells and extracellular matrix proteins induces mRNA expression of both the mitogen-activated protein (MAP) kinase P. carinii Ste20 (PcSte20) and the cell wall-remodeling enzyme PcCbk1 (16). Herein, we report that in addition to PcSte20 mRNA expression being upregulated, Pneumocystis PcSte20 kinase activity is increased upon interacting with these same lung targets. This activity is also significantly suppressed by Clostridium difficile toxin B, a pan-specific inhibitor of small GTPases, demonstrating the potential role of a Cdc42-like molecule in this signaling cascade. We further observed that the PcSte20 kinase physically interacts with a specific region of the P. carinii cell wall biosynthesis kinase, PcCbk1, a downstream kinase important for mating projection formation and cell wall remodeling. This direct binding was mapped to a specific region of the PcCbk1 protein. We also demonstrated that PcSte20 obtained from whole P. carinii lysates has the ability to phosphorylate PcCbk1 after the organism interacts with lung epithelial cells and extracellular matrix components. These observations provide new insights into P. carinii signaling induced by interactions of this important opportunistic fungal pathogen with lung epithelial cells and matrix.