1 Scientific RepoRts | 6:23927 | DOI: 10.1038/srep23927 www.nature.com/scientificreports Identifcation of a major IP 5 kinase in Cryptococcus neoformans confrms that PP-IP 5 /IP 7 , not IP 6 , is essential for virulence Cecilia Li 1 , Sophie Lev 1 , Adolfo Saiardi 2 , Desmarini Desmarini 1 , Tania C. Sorrell 1,3,4 & Julianne T. Djordjevic 1,3,4 Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP 3 to inositol pyrophosphate, PP-IP 5 /IP 7 , which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP 6 to PP-IP 5 /IP 7 , but the kinase converting IP 5 to IP 6 is unknown. Deletion of a putative IP 5 kinase- encoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1Δkcs1Δ and kcs1Δ lung burdens were also lower than those of ipk1Δ. Unlike ipk1Δ, ipk1Δkcs1Δ and kcs1Δ failed to disseminate to the brain. IP profling confrmed Ipk1 as the major IP 5 kinase in C. neoformans: ipk1Δ produced no IP 6 or PP-IP 5 /IP 7 and, in contrast to ipk1Δkcs1Δ, accumulated IP 5 and its pyrophosphorylated PP-IP 4 derivative. Kcs1 is therefore a dual specifcity (IP 5 and IP 6 ) kinase producing PP-IP 4 and PP-IP 5 /IP 7 . All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced signifcantly in all mutants except ipk1Δ, suggesting that PP-IP 4 partially compensates for absent PP-IP 5 /IP 7 in ipk1Δ grown under this condition. In conclusion, PP-IP 5 / IP 7 , not IP 6 , is essential for fungal virulence. Te fungal pathogen, Cryptococcus neoformans, predominantly infects immunocompromised individuals via the lung and then disseminates to the brain where it establishes life-threatening meningoencephalitis. C. neoformans is responsible for over half a million deaths each year in AIDS patients alone 1 . Some of the most well-known virulence factors of C. neoformans include a polysaccharide capsule 2 , melanin 3 and urease 4,5 . A prerequisite to vir- ulence is its ability to grow at human physiological temperature, which can impact the stability of the cell wall 6,7 . In the host, C. neoformans also encounters oxidative and nitrosative stress, which originate predominantly from macrophages and altered nutritional availability in the lung and brain, which are low in glucose 8–13 . Several signalling cascades, including calcineurin, mitogen-activated protein kinase/protein kinase C (Mpk1/ Pkc1), cyclic adenosine monophosphate/protein kinase A (cAMP/Pka1), high osmolarity glycerol (HOG), and Rim101 pathways, allow C. neoformans to sense, respond and adapt to host stresses encountered throughout the course of infection 14–20 . We previously identifed a new virulence-related signalling pathway in C. neoformans comprising phospholipase C1 (Plc1) and a series of sequentially acting inositol polyphosphate kinases (IPKs) 21–23 . Te IPKs convert the inositol trisphosphate (IP 3 ) product of Plc1 to IP 4 -IP 6 , and IP 6 to the inositol pyrophos- phates, PP-IP 5 /IP 7 and (PP) 2 -IP 4 /IP 8 . Specifcally, IP 3 is converted to IP 4 and IP 5 by Arg1 22,23 . An uncharacterised kinase then phosphorylates IP 5 to produce the highly abundant IP 6 species. IP 6 is then phosphorylated by Kcs1 to PP-IP 5 /IP 7 . Asp1 further phosphorylates PP-IP 5 /IP 7 to produce (PP) 2 -IP 4 /IP 8 23 . ARG1 and KCS1 deletion mutants of C. neoformans, which do not produce PP-IP 5 /IP 7 and (PP) 2 -IP 4 /IP 8 , exhibit attenuated growth, compromised cell wall integrity and reduced production of melanin, urease and mating flaments. Te KCS1 deletion strain, kcs1Δ, is also unable to utilise alternative carbon sources for growth 23 . Consequently kcs1Δ has a reduced ability 1 centre for infectious Diseases and Microbiology, the Westmead institute for Medical Research, the University of Sydney, Westmead, nSW, Australia. 2 Medical Research council Laboratory for Molecular cell Biology, University college London, London, UK. 3 Marie Bashir institute for infectious Diseases and Biosecurity, University of Sydney, nSW, Australia. 4 Westmead Hospital, Westmead, nSW, Australia. correspondence and requests for materials should be addressed to J. t.D. (email: julianne.djordjevic@sydney.edu.au) Received: 22 January 2016 Accepted: 15 March 2016 Published: 01 April 2016 OPEN