Sophie LevDesmarini DesmariniCecilia LiMethee ChayakulkeereeAna TravenTania C. SorrellJulianne T. DjordjevicUniversity of Sydney Faculty of MedicineMahidol UniversityMonash UniversityThe University of Sydney2018-10-192018-10-192013-04-01Infection and Immunity. Vol.81, No.4 (2013), 1245-125510985522001995672-s2.0-84875544623https://repository.li.mahidol.ac.th/handle/123456789/31940Phospholipase C (PLC) of Cryptococcus 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). In Saccharomyces cerevisiae, Plc1-derived IP3 is a substrate for the inositol polyphosphate kinase Arg82, which converts IP3 to more complex inositol polyphosphates. In this study, we show that in C. neoformans, the enzyme encoded by ARG1 is the major IP3 kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in the Galleria mellonella infection model. IP3 content was reduced in the Cnδplc1 mutant and markedly increased in the Cnδarg1 mutant, while PIP2 was increased in both mutants. The Cnδplc1 and Cnδarg1 mutants 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 the S. cerevisiae ARG82 deletion mutant (Scδarg82) strain, the Cnδarg1 mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP3 causes 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 of C. neoformans and further show that production of IP3 as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1. © 2013, American Society for Microbiolog.Mahidol UniversityImmunology and MicrobiologyMedicineArticleSCOPUS10.1128/IAI.01421-12