IP₇-SPX Domain Interaction Controls Fungal Virulence by Stabilizing Phosphate Signaling Machinery

In the human-pathogenic fungus Cryptococcus neoformans, the inositol polyphosphate signaling pathway is critical for virulence. We recently demonstrated the key role of the inositol pyrophosphate IP₇ (isomer 5-PP-IP₅) in driving fungal virulence; however, the mechanism of action remains elusive. Using genetic and biochemical approaches, and mouse infection models, we show that IP₇ synthesized by Kcs1 regulates fungal virulence by binding to a conserved lysine surface cluster in the SPX domain of Pho81. Pho81 is the cyclin-dependent kinase (CDK) inhibitor of the phosphate signaling (PHO) pathway. We also provide novel mechanistic insight into the role of IP₇ in PHO pathway regulation by demonstrating that IP₇ functions as an intermolecular "glue" to stabilize Pho81 association with Pho85/Pho80 and, hence, promote PHO pathway activation and phosphate acquisition. Blocking IP₇-Pho81 interaction using site-directed mutagenesis led to a dramatic loss of fungal virulence in a mouse infection model, and the effect was similar to that observed following PHO81 gene deletion, highlighting the key importance of Pho81 in fungal virulence. Furthermore, our findings provide additional evidence of evolutionary divergence in PHO pathway regulation in fungi by demonstrating that IP₇ isomers have evolved different roles in PHO pathway control in C. neoformans and nonpathogenic yeast.IMPORTANCE Invasive fungal diseases pose a serious threat to human health globally with >1.5 million deaths occurring annually, 180,000 of which are attributable to the AIDS-related pathogen, Cryptococcus neoformans Here, we demonstrate that interaction of the inositol pyrophosphate, IP₇, with the CDK inhibitor protein, Pho81, is instrumental in promoting fungal virulence. IP₇-Pho81 interaction stabilizes Pho81 association with other CDK complex components to promote PHO pathway activation and phosphate acquisition. Our data demonstrating that blocking IP₇-Pho81 interaction or preventing Pho81 production leads to a dramatic loss in fungal virulence, coupled with Pho81 having no homologue in humans, highlights Pho81 function as a potential target for the development of urgently needed antifungal drugs.