Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization

ABSTRACT Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that c...

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Autores principales:	Giuseppe Ianiri, Alexander Idnurm
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9910993961724cf7b61564e6653c68cb
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spelling	oai:doaj.org-article:9910993961724cf7b61564e6653c68cb2021-11-15T15:41:34ZEssential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization10.1128/mBio.02334-142150-7511https://doaj.org/article/9910993961724cf7b61564e6653c68cb2015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02334-14https://doaj.org/toc/2150-7511ABSTRACT Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in both Cryptococcus and ascomycete human pathogens would be ideal for the development of antifungal drugs with broad-spectrum activity. IMPORTANCE Fungal infections are very common in humans but may be neglected due to misdiagnosis and inattention. Cryptococcus neoformans is a yeast that infects mainly immunocompromised people, causing high mortality rates in developing countries. The fungus infects the lungs, crosses the blood-brain barrier, and invades the cerebrospinal fluid, causing fatal meningitis. C. neoformans infections are treated with amphotericin B, flucytosine, and azoles, all developed decades ago. However, problems with antifungal agents highlight the urgent need for more-effective drugs to treat C. neoformans and other invasive fungal infections. These issues include the negative side effects of amphotericin B, the spontaneous resistance of C. neoformans to azoles, and the inefficacy of the echinocandin antifungals. In this study, we report the identification of C. neoformans essential genes as targets for the development of novel antifungals. Because of the level of evolutionary divergence between C. neoformans and the ascomycetes, a subset of these genes is likely essential in all fungi. Genes identified in this study represent an excellent starting point for the future development of new antifungals by pharmaceutical companies.Giuseppe IaniriAlexander IdnurmAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Giuseppe Ianiri Alexander Idnurm Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization
description	ABSTRACT Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in both Cryptococcus and ascomycete human pathogens would be ideal for the development of antifungal drugs with broad-spectrum activity. IMPORTANCE Fungal infections are very common in humans but may be neglected due to misdiagnosis and inattention. Cryptococcus neoformans is a yeast that infects mainly immunocompromised people, causing high mortality rates in developing countries. The fungus infects the lungs, crosses the blood-brain barrier, and invades the cerebrospinal fluid, causing fatal meningitis. C. neoformans infections are treated with amphotericin B, flucytosine, and azoles, all developed decades ago. However, problems with antifungal agents highlight the urgent need for more-effective drugs to treat C. neoformans and other invasive fungal infections. These issues include the negative side effects of amphotericin B, the spontaneous resistance of C. neoformans to azoles, and the inefficacy of the echinocandin antifungals. In this study, we report the identification of C. neoformans essential genes as targets for the development of novel antifungals. Because of the level of evolutionary divergence between C. neoformans and the ascomycetes, a subset of these genes is likely essential in all fungi. Genes identified in this study represent an excellent starting point for the future development of new antifungals by pharmaceutical companies.
format	article
author	Giuseppe Ianiri Alexander Idnurm
author_facet	Giuseppe Ianiri Alexander Idnurm
author_sort	Giuseppe Ianiri
title	Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization
title_short	Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization
title_full	Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization
title_fullStr	Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization
title_full_unstemmed	Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization
title_sort	essential gene discovery in the basidiomycete <named-content content-type="genus-species">cryptococcus neoformans</named-content> for antifungal drug target prioritization
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/9910993961724cf7b61564e6653c68cb
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Essential Gene Discovery in the Basidiomycete <named-content content-type="genus-species">Cryptococcus neoformans</named-content> for Antifungal Drug Target Prioritization

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