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

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 (ipk1?), and in combination with KCS1 (ipk1?kcs1?), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1?kcs1? and kcs1? lung burdens were also lower than those of ipk1?. Unlike ipk1?, ipk1?kcs1? and kcs1? failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1? produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1?kcs1?, 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 ipk1?, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1? grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence.

SUBMITTER: Li C

PROVIDER: S-EPMC4817067 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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

Li Cecilia C Lev Sophie S Saiardi Adolfo A Desmarini Desmarini D Sorrell Tania C TC Djordjevic Julianne T JT

Scientific reports 20160401

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 attenuatio ...[more]

PMID: 27033523

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Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

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Dataset Information

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

Publications

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

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