Phospholipase C of Cryptococcus neoformans Regulates Homeostasis and Virulence by Providing Inositol Trisphosphate as a Substrate for Arg1 Kinase

Abstract

ABSTRACTPhospholipase C (PLC) ofCryptococcus neoformans(CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP2) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP3). InSaccharomyces cerevisiae, Plc1-derived IP3is a substrate for the inositol polyphosphate kinase Arg82, which converts IP3to more complex inositol polyphosphates. In this study, we show that inC. neoformans, the enzyme encoded byARG1is the major IP3kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in theGalleria mellonellainfection model. IP3content was reduced in the CnΔplc1mutant and markedly increased in the CnΔarg1mutant, while PIP2was increased in both mutants. The CnΔplc1and CnΔarg1mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to theS. cerevisiae ARG82deletion mutant (ScΔarg82) strain, the CnΔarg1mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP3causes Ca2+release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence ofC. neoformansand further show that production of IP3as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1.