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Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin

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Abstract

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding β-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of β-1, 3-glucan synthesis in C. neoformans; the inhibition of β-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized β-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

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Acknowledgements

This study was supported by the university fund from Chiba-University. Micafungin was provided by Astellas Pharma Inc., Japan. There is no conflict of interest.

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Authors and Affiliations

  1. Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chiba, 260-8673, Japan

    Akio Toh-e, Misako Ohkusu, Masashi Yamaguchi, Naruhiko Ishiwada, Akira Watanabe & Katsuhiko Kamei

  2. Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan

    Kiminori Shimizu

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  1. Akio Toh-e
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Correspondence to Akio Toh-e.

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Communicated by M. Kupiec.

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Toh-e, A., Ohkusu, M., Shimizu, K. et al. Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin. Curr Genet 63, 1093–1104 (2017). https://doi.org/10.1007/s00294-017-0713-8

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