Skip to main content

Advertisement

SpringerLink
Log in

Isolation of conditional mutations in genes essential for viability of Cryptococcus neoformans

  • Original Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Discovering the genes underlying fundamental processes that enable cells to live and reproduce is a technical challenge, because loss of gene function in mutants results in organisms that cannot survive. This study describes a forward genetics method to identify essential genes in fungi, based on the propensity for Agrobacterium tumefaciens to insert T-DNA molecules into the promoters or 5′ untranslated regions of genes and by placing a conditional promoter within the T-DNA. Insertions of the promoter of the GAL7 gene were made in the human pathogen Cryptococcus neoformans. Nine strains of 960 T-DNA insertional mutants screened grew on media containing galactose, but had impaired growth on media containing glucose, which suppresses expression from GAL7. T-DNA insertions were found in the homologs of IDI1, MRPL37, NOC3, NOP56, PRE3 and RPL17, all of which are essential in ascomycete yeasts Saccharomyces cerevisiae or Schizosaccharomyces pombe. Altering the carbon source in the medium provided a system to identify phenotypes in response to stress agents. The pre3 proteasome subunit mutant was further characterized. The T-DNA insertion and phenotype co-segregate in progeny from a cross, and the growth defect is complemented by the reintroduction of the wild type gene into the insertional mutant. A deletion allele was generated in a diploid strain, this heterozygous strain was sporulated, and analysis of the progeny provided additional genetic evidence that PRE3 is essential. The experimental design is applicable to other fungi and has other forward genetic applications such as to isolate over-expression suppressors or enhance the production of traits of interest.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Baker LG, Lodge JK (2012) Galactose-inducible promoters in Cryptococcus neoformans var. grubii. Methods Mol Biol 845:211–226

    Article  CAS  PubMed  Google Scholar 

  • Becker JM, Kauffman SJ, Hauser M, Huang L, Lin M, Sillaots S, Jiang B, Xu D, Roemer T (2010) Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model. Proc Natl Acad Sci USA 107:22044–22049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Berbee ML, Taylor JW (2010) Dating the molecular clock in fungi—how close are we? Fungal Biol Rev 24:1–16

    Article  Google Scholar 

  • Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214

    CAS  PubMed  PubMed Central  Google Scholar 

  • Calderone R, Sun N, Gay-Andrieu F, Groutas W, Weerawarna P, Prasad S, Alex D, Li D (2014) Antifungal drug discovery: the process and outcomes. Future Microbiol 9:791–805

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chambers K, Lowe RGT, Howlett BJ, Zander M, Batley J, Van de Wouw AP, Elliott CE (2014) Next-generation genome sequencing can be used to rapidly characterise sequences flanking T-DNA insertions in random insertional mutants of Leptosphaeria maculans. Fungal Biol Biotechnol 1:10

    Article  Google Scholar 

  • Covert SF, Kapoor P, Lee M-H, Briley A, Nairn CJ (2001) Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol Res 105:259–264

    Article  CAS  Google Scholar 

  • Del Poeta M, Toffaletti DL, Rude TH, Dykstra CC, Heitman J, Perfect JR (1999) Topoisomerase I is essential in Cryptococcus neoformans: role in pathobiology and as an antifungal target. Genetics 152:167–178

    PubMed  PubMed Central  Google Scholar 

  • Denning DW, Bromley MJ (2015) How to bolster the antifungal pipeline. Science 347:1414–1416

    Article  CAS  PubMed  Google Scholar 

  • Dowell RD, Ryan O, Jansen A, Cheung D, Agarwala S, Danford T, Bernstein DA, Rolfe PA, Heisler LE, Chin B, Nislow C, Giaever G, Phillips PC, Fink GR, Gifford DK, Boone C (2010) Genotype to phenotype: a complex problem. Science 328:469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Enenkel C, Lehmann H, Kipper J, Gückel R, Hilt W, Wolf DH (1994) PRE3, highly homologous to the human major histocompatibility complex-linked LMP2 (RING12) gene, codes for a yeast proteasome subunit necessary for the peptidylglutamyl-peptide hydrolyzing activity. FEBS Lett 341:193–196

    Article  CAS  PubMed  Google Scholar 

  • Esher SK, Granek JA, Alspaugh JA (2015) Rapid mapping of insertional mutations to probe cell wall regulation in Cryptococcus neoformans. Fungal Genet Biol 82:9–21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Firon A, Villalba F, Beffa R, d’Enfert C (2003) Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis. Eukaryot Cell 2:247–255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gamalinda M, Jakovljevic J, Babiano R, Talkish J, de la Cruz J, Woolford JL Jr (2013) Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing. Nucleic Acids Res 41:1965–1983

    Article  CAS  PubMed  Google Scholar 

  • Gautier T, Bergès T, Tollervey D, Hurt E (1997) Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis. Mol Cell Biol 17:7088–7098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, Dow S, Lucau-Danila A, Anderson K, André B, Arkin AP, Astromoff A, El-Bakkoury M, Bangham R, Benito R, Brachat S, Campanaro S, Curtiss M, Davis K, Deutschbauer A, Entian K-D, Flaherty P, Foury F, Garfinkel DJ, Gerstein M, Gotte D, Güldener U, Hegemann JH, Hempel S, Herman Z, Jaramillo DF, Kelly DE, Kelly SL, Kötter P, LaBonte D, Lamb DC, Lan N, Liang H, Liao H, Liu L, Luo C, Lussier M, Mao R, Menard P, Ooi SL, Revuelta JL, Roberts CJ, Rose M, Ross-Macdonald P, Scherens B, Schimmack G, Shafer B, Shoemaker DD, Sookhai-Mahadeo S, Storms RK, Strathern JN, Valle G, Voet M, Volckaert G, Wang C-Y, Ward TR, Wilhelmy J, Winzeler EA, Yang Y, Yen G, Youngman E, Yu K, Bussey H, Boeke JD, Snyder M, Philippsen P, Davis RW, Johnston M (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391

    Article  CAS  PubMed  Google Scholar 

  • Hagen F, Khayhan K, Theelen B, Kolecka A, Polacheck I, Sionov E, Falk R, Parnmen S, Lumbsch HT, Boekhout T (2015) Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol 78:16–48

    Article  CAS  PubMed  Google Scholar 

  • Hanlon SE, Xu Z, Norris DN, Vershon AK (2004) Analysis of the meiotic role of the mitochondrial ribosomal proteins Mrps17 and Mrpl37 in Saccharomyces cerevisiae. Yeast 21:1241–1252

    Article  CAS  PubMed  Google Scholar 

  • Heinemeyer W, Fischer M, Krimmer T, Stachon U, Wolf DH (1997) The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing. J Biol Chem 272:25200–25209

    Article  CAS  PubMed  Google Scholar 

  • Homer CM, Summers DK, Goranov AI, Clarke SC, Wiesner DL, Diedrich JK, Moresco JJ, Toffaletti D, Upadhya R, Caradonna I, Petnic S, Pessino V, Cuomo CA, Lodge JK, Perfect J, Yates JR 3rd, Nielsen K, Craik CS, Madhani HD (2016) Intracellular action of a secreted peptide required for fungal virulence. Cell Host Microbe 19:849–864

    Article  CAS  PubMed  Google Scholar 

  • Houchens CR, Perreault A, Bachand F, Kelly TJ (2008) Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication. Eukaryot Cell 7:1433–1440

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu W, Sillaots S, Lemieux S, Davison J, Kauffman S, Breton A, Linteau A, Xin C, Bowman J, Becker J, Jiang B, Roemer T (2007) Essential gene identification and drug target prioritization in Aspergillus fumigatus. Plos Pathog 3:e24

    Article  PubMed  PubMed Central  Google Scholar 

  • Ianiri G, Idnurm A (2015) Essential gene discovery in the basidiomycete Cryptococcus neoformans for antifungal drug target prioritization. mBio 6:e02334

    Article  PubMed  PubMed Central  Google Scholar 

  • Idnurm A (2010) A tetrad analysis of the basidiomycete fungus Cryptococcus neoformans. Genetics 185:153–163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Idnurm A, Lin X (2015) Rising to the challenge of multiple Cryptococcus species and the diseases they cause. Fungal Genet Biol 78:1–6

    Article  PubMed  PubMed Central  Google Scholar 

  • Idnurm A, Reedy JL, Nussbaum JC, Heitman J (2004) Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell 3:420–429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    CAS  PubMed  PubMed Central  Google Scholar 

  • Janbon G, Ormerod KL, Paulet D, Byrnes EJ 3rd, Yadav V, Chatterjee G, Mullapudi N, Hon C-C, Billmyre RB, Brunel F, Bahn Y-S, Chen W, Chen Y, Chow EWL, Coppée J-Y, Floyd-Averette A, Gaillardin C, Gerik KJ, Goldberg J, Gonzalez-Hilarion S, Gujja S, Hamlin JL, Hsueh Y-P, Ianiri G, Jones S, Kodira CD, Kozubowski L, Lam W, Marra M, Mesner LD, Mieczkowski PA, Moyrand F, Nielsen K, Proux C, Rossignol T, Schein JE, Sun S, Wollschlaeger C, Wood IA, Zeng Q, Neuvéglise C, Newlon CS, Perfect JR, Lodge JK, Idnurm A, Stajich JE, Kronstad JW, Sanyal K, Heitman J, Fraser JA, Cuomo CA, Dietrich FS (2014) Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. Plos Genet 10:e1004261

    Article  PubMed  PubMed Central  Google Scholar 

  • Kim D-U, Hayles J, Kim D, Wood V, Park H-O, Won M, Yoo H-S, Duhig T, Nam M, Palmer G, Han S, Jeffery L, Baek S-T, Lee H, Shim YS, Lee M, Kim L, Heo K-S, Noh EJ, Lee A-R, Jang Y-J, Chung K-S, Choi S-J, Park J-Y, Park Y, Kim HM, Park S-K, Park H-J, Kang E-J, Kim HB, Kang H-S, Park H-M, Kim K, Song K, Song KB, Nurse P, Hoe K-L (2010) Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 28:617–623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Krysan DJ (2015) Toward improved anti-cryptococcal drugs: novel molecules and repurposed drugs. Fungal Genet Biol. 78:93–98

    Article  CAS  PubMed  Google Scholar 

  • Lai W-C, Sun H-FS, Lin P-H, Lin H, Shieh J-C (2016) A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans. Curr Genet 62:213–235

    Article  CAS  PubMed  Google Scholar 

  • Liu OW, Chun CD, Chow ED, Chen C, Madhani HD, Noble SM (2008) Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135:174–188

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luberto C, Toffaletti DL, Wills EA, Tucker SC, Casadevall A, Perfect JR, Hannun YA, Del Poeta MM (2001) Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans. Genes Dev 15:201–212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mayer MP, Hahn FM, Stillman DJ, Poulter CD (1992) Disruption and mapping of IDI1, the gene for isopentenyl diphosphate isomerase in Saccharomyces cerevisiae. Yeast 8:743–748

    Article  CAS  PubMed  Google Scholar 

  • Merz S, Westermann B (2009) Genome-wide deletion mutant analysis reveals genes required for respiratory growth, mitochondrial genome maintenance and mitochondrial protein synthesis in Saccharomyces cerevisiae. Genome Biol 10:R95

    Article  PubMed  PubMed Central  Google Scholar 

  • Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet 48:1–17

    Article  CAS  PubMed  Google Scholar 

  • Milkereit P, Gadal O, Podtelejnikov A, Trumtel S, Gas N, Petfalski E, Tollervey D, Mann M, Hurt E, Tschochner H (2001) Maturation and intranuclear transport of pre-ribosomes requires Noc proteins. Cell 105:499–509

    Article  CAS  PubMed  Google Scholar 

  • Nielsen K, Cox GM, Wang P, Toffaletti DL, Perfect JR, Heitman J (2003) Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and α isolates. Infect Immun 71:4831–4841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Okuda T, Ando A, Sakuradani E, Kikukawa H, Kamada N, Ochiai M, Shima J, Ogawa J (2014) Characterization of galactose-dependent promoters from an oleaginous fungus Mortierella alpina 1S-4. Curr Genet 60:175–182

    Article  CAS  PubMed  Google Scholar 

  • Ory JJ, Griffith CL, Doering TL (2004) An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter. Yeast 21:919–926

    Article  CAS  PubMed  Google Scholar 

  • Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23:525–530

    Article  PubMed  Google Scholar 

  • Park J, Rajasingham R, Smith RM, Boulware DR (2014) Update on the global burden of cryptococcosis. Mycoses 57S1:6

    Google Scholar 

  • Pitkin JW, Panaccione DG, Walton JD (1996) A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum. Microbiology 142:1557–1565

    Article  CAS  PubMed  Google Scholar 

  • Roemer T, Krysan DJ (2014) Antifungal drug development: challenges, unmet clinical needs, and new approaches. Cold Spring Harb Perspect Med 4:a019703

    Article  PubMed  PubMed Central  Google Scholar 

  • Ruff JA, Lodge JK, Baker LG (2009) Three galactose inducible promoters for use in C. neoformans var. grubii. Fungal Genet Biol 46:9–16

    Article  CAS  PubMed  Google Scholar 

  • Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27

    CAS  PubMed  PubMed Central  Google Scholar 

  • Taylor JW, Berbee ML (2006) Dating divergences in the fungal tree of life: review and new analyses. Mycologia 98:838–849

    Article  PubMed  Google Scholar 

  • Toffaletti DL, Rude TH, Johnston SA, Durack DT, Perfect JR (1993) Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175:1405–1411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walton FJ, Idnurm A, Heitman J (2005) Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol Microbiol 57:1381–1396

    Article  CAS  PubMed  Google Scholar 

  • Wickes BL, Edman JC (1995) The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter. Mol Microbiol 16:1099–1109

    Article  CAS  PubMed  Google Scholar 

  • Winston F, Dollard C, Ricupero-Hovasse SL (1995) Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11:53–55

    Article  CAS  PubMed  Google Scholar 

  • Xue C, Tada Y, Dong X, Heitman J (2007) The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants. Cell Host Microbe 1:263–273

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Yu Z, Fu X, Liang C (2002) Noc3p, a bHLH protein, plays an integral role in the initiation of DNA replication in budding yeast. Cell 109:849–860

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was initiated at the University of Missouri-Kansas City: from there we thank Colleen Mayhue, Chris Naumann and Nghi Chau for their technical help. The work was supported by grants from the United States National Institutes of Health (AI094364) and Australian Research Council (FT130100146).

Author information

Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, 27710, USA

    Giuseppe Ianiri

  2. Dipartimento di Agricoltura, Ambiente e Alimenti, Università degli Studi del Molise, Via F. De Sanctis Snc, 86100, Campobasso, Italy

    Giuseppe Ianiri

  3. School of BioSciences, BioSciences 2, University of Melbourne, Building 122, Melbourne, VIC, 3010, Australia

    Kylie J. Boyce & Alexander Idnurm

Authors
  1. Giuseppe Ianiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kylie J. Boyce
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexander Idnurm
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexander Idnurm.

Additional information

Communicated by M. Kupiec.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ianiri, G., Boyce, K.J. & Idnurm, A. Isolation of conditional mutations in genes essential for viability of Cryptococcus neoformans . Curr Genet 63, 519–530 (2017). https://doi.org/10.1007/s00294-016-0659-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-016-0659-2

Keywords

Search

Navigation