title: Essential gene discovery in the basidiomycete Cryptococcus neoformans 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 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.
  
  
  OBJECTIVE
  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.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ianiri
authors.affiliation: alexander.idnurm@unimelb.edu.au.
authors.forename: Alexander
authors.initials: A
authors.lastname: Idnurm
mesh_terms: D000935:Antifungal Agents / Q000302:isolation & purification*; D003455:Cryptococcus neoformans / Q000235:genetics*; D055808:Drug Discovery / Q000379:methods; D020043:Genes, Essential; D005800:Genes, Fungal; D005827:Genetics, Microbial / Q000379:methods*; D050296:Microbial Viability; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional
chemical_list: D000935:Antifungal Agents
references.citation: Mycobiology. 2010 Sep;38(3):215-8
references.pmid: 23956658
references.citation: Mycologia. 2006 Nov-Dec;98(6):838-49
references.pmid: 17486961
references.citation: Chem Biol. 2011 Feb 25;18(2):148-64
references.pmid: 21338914
references.citation: Eukaryot Cell. 2008 Apr;7(4):619-29
references.pmid: 18245278
references.citation: PLoS Genet. 2005 Jul;1(1):36-57
references.pmid: 16103911
references.citation: Eukaryot Cell. 2003 Apr;2(2):247-55
references.pmid: 12684374
references.citation: Mol Microbiol. 2001 May;40(3):610-20
references.pmid: 11359567
references.citation: Antimicrob Agents Chemother. 1999 Aug;43(8):1856-61
references.pmid: 10428902
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D675-81
references.pmid: 22064857
references.citation: Science. 2010 Apr 23;328(5977):469
references.pmid: 20413493
references.citation: PLoS Genet. 2014 Apr 17;10(4):e1004261
references.pmid: 24743168
references.citation: Mol Biol Cell. 2003 Sep;14(9):3782-803
references.pmid: 12972564
references.citation: Mycoses. 2014 Dec;57(12):741-6
references.pmid: 25135153
references.citation: J Biol Chem. 1988 Dec 5;263(34):18443-51
references.pmid: 2903861
references.citation: PLoS Genet. 2010 Oct 14;6(10):e1001154
references.pmid: 20976243
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):793-8
references.pmid: 14718668
references.citation: Mol Biol Cell. 2006 Sep;17(9):3768-80
references.pmid: 16775005
references.citation: PLoS Pathog. 2012;8(10):e1002936
references.pmid: 23055925
references.citation: PLoS One. 2011 Feb 28;6(2):e16719
references.pmid: 21386897
references.citation: PLoS Genet. 2012 Sep;8(9):e1002950
references.pmid: 23028357
references.citation: Eukaryot Cell. 2010 Mar;9(3):438-48
references.pmid: 20097738
references.citation: Sci Transl Med. 2012 Dec 19;4(165):165rv13
references.pmid: 23253612
references.citation: J Biol Chem. 2015 Jan 16;290(3):1861-73
references.pmid: 25477510
references.citation: Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):22044-9
references.pmid: 21135205
references.citation: J Biol Chem. 2005 Dec 2;280(48):39835-42
references.pmid: 16204227
references.citation: Eukaryot Cell. 2005 Feb;4(2):487-9
references.pmid: 15701811
references.citation: Nat Rev Drug Discov. 2010 Sep;9(9):719-27
references.pmid: 20725094
references.citation: Infect Immun. 2003 Sep;71(9):4831-41
references.pmid: 12933823
references.citation: PLoS One. 2013 Sep 03;8(9):e74737
references.pmid: 24019979
references.citation: Emerg Infect Dis. 2009 Aug;15(8):1185-91
references.pmid: 19757550
references.citation: PLoS Pathog. 2010 Apr 22;6(4):e1000850
references.pmid: 20421942
references.citation: Mol Biol Cell. 2008 Oct;19(10):4310-8
references.pmid: 18667528
references.citation: PLoS One. 2010 Apr 13;5(4):e10168
references.pmid: 20405015
references.citation: J Bacteriol. 1993 Mar;175(5):1405-11
references.pmid: 8444802
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10352-10357
references.pmid: 16801547
references.citation: Clin Microbiol Rev. 2005 Jan;18(1):163-94
references.pmid: 15653825
references.citation: Trends Microbiol. 2002 Oct;10(10):456-62
references.pmid: 12377555
references.citation: PLoS Pathog. 2007 Mar;3(3):e24
references.pmid: 17352532
references.citation: Mol Microbiol. 2003 Oct;50(1):167-81
references.pmid: 14507372
references.citation: Mol Microbiol. 2005 Sep;57(5):1381-96
references.pmid: 16102007
references.citation: Genes Dev. 1999 Aug 15;13(16):2148-58
references.pmid: 10465791
references.citation: Nat Biotechnol. 2010 Jun;28(6):617-623
references.pmid: 20473289
references.citation: Science. 2009 Mar 27;323(5922):1693-7
references.pmid: 19325107
references.citation: Curr Opin Microbiol. 2010 Dec;13(6):663-71
references.pmid: 20869909
references.citation: Value Health. 2002 Jan-Feb;5(1):26-34
references.pmid: 11873380
references.citation: Eukaryot Cell. 2005 Jun;4(6):1079-87
references.pmid: 15947200
references.citation: Eukaryot Cell. 2004 Apr;3(2):420-9
references.pmid: 15075272
references.citation: PLoS Pathog. 2010 Oct 07;6(10):e1001140
references.pmid: 20949076
references.citation: Science. 1999 Aug 6;285(5429):901-6
references.pmid: 10436161
references.citation: EMBO J. 1998 Jul 1;17(13):3597-607
references.pmid: 9649430
references.citation: Biochem Biophys Res Commun. 1997 Oct 9;239(1):116-22
references.pmid: 9345280
references.citation: Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13227-32
references.pmid: 11078525
references.citation: Eukaryot Cell. 2009 Mar;8(3):315-26
references.pmid: 19151325
references.citation: Cell Host Microbe. 2007 Jun 14;1(4):263-73
references.pmid: 18005707
references.citation: Eukaryot Cell. 2014 Jul;13(7):878-83
references.pmid: 24813190
references.citation: BMC Genomics. 2011 Jan 27;12:75
references.pmid: 21272313
references.citation: PLoS Pathog. 2010 Apr 01;6(4):e1000848
references.pmid: 20368972
references.citation: Cell. 2008 Oct 3;135(1):174-88
references.pmid: 18854164
references.citation: J Bacteriol. 1999 Jan;181(2):444-53
references.pmid: 9882657
references.citation: Genetics. 2010 May;185(1):153-63
references.pmid: 20157004
references.citation: Nat Genet. 1999 Mar;21(3):278-83
references.pmid: 10080179
references.citation: PLoS Pathog. 2012 Sep;8(9):e1002870
references.pmid: 22969422
references.citation: PLoS Pathog. 2007 Jun;3(6):e92
references.pmid: 17604452
references.citation: mBio. 2015 Mar 31;6(2):
references.pmid: 25827419
references.citation: PLoS Biol. 2013 Sep;11(9):e1001653
references.pmid: 24058295
references.citation: J Cell Biol. 2000 Oct 16;151(2):263-76
references.pmid: 11038174
references.citation: AIDS. 2009 Feb 20;23(4):525-30
references.pmid: 19182676