Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence.

Cecilia Li,Tania C Sorrell,Sophie Lev,Julianne T Djordjevic,Desmarini Desmarini,Adolfo Saiardi

doi:10.1038/srep23927

Abstract

Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 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 profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. Kcs1 is therefore a dual specificity (IP5 and IP6) kinase producing PP-IP4 and PP-IP5/IP7. All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced significantly in all mutants except ipk1Δ, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1Δ grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence.

Highlights

Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7 and (PP)-IP5/IP7, which is essential for virulence of Cryptococcus neoformans
Similar to Kcs[1] we found that Ipk[1] is essential for pathogenicity but that its contribution relates more to its indirect role in PP-IP5/IP7 production, rather than its direct role in producing IP6
We identified Kcs[1] as the major cryptococcal IP6 kinase, phosphorylating IP6 to the inositol pyrophosphate PP-IP5/IP7, which is crucial for cryptococcal virulence[23]

Summary

Introduction

Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Deletion of a putative IP5 kinaseencoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. IP profiling confirmed Ipk[1] as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. All mutants were attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. ARG1 and KCS1 deletion mutants of C. neoformans, which do not produce PP-IP5/IP7 and (PP)2-IP4/IP8, exhibit attenuated growth, compromised cell wall integrity and reduced production of melanin, urease and mating filaments. The KCS1 deletion strain, kcs1Δ, is unable to utilise alternative carbon sources for growth[23].

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Conclusion