Arg1 from lacks PI3 kinase activity and conveys virulence roles via its IP kinase activity.

Inositol tris/tetrakis phosphate kinases (IPK) in the human fungal priority pathogens, (Arg1) and (Ipk2), convey numerous virulence functions, yet it is not known whether the IPK catalytic activity or a scaffolding role is responsible. We therefore generated a strain with a non-functional kinase, referred to as the dead-kinase (dk) Arg1 strain (dkArg1). We verified that, although dk cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IPK activity. Using recombinant Arg1 and Ipk2 we confirmed that, unlike the IPK homologs in humans and , Arg1 and Ipk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP conversion is blocked in the dkArg1 and deletion (Δ) strains and that 1-IP and a recently discovered isomer (4/6-IP) are made by wild-type . Importantly, the dkArg1 and Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IPK catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to since Arg1 is dispensable for growth on different nitrogen sources. IPK catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IPK is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, and , and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IPK enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IPK catalytic activity of Arg1 promotes all virulence traits in that are attenuated by deletion reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IPK catalytic site, as a new antifungal drug class.