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

Abstract

In the human-pathogenic fungus <i>Cryptococcus neoformans</i>, 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 <i>PHO81</i> 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 <i>C. neoformans</i> and nonpathogenic yeast.<b>IMPORTANCE</b> 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, <i>Cryptococcus neoformans</i> 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.