Current Genetics

, 49:341

Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition

Technical Note

Abstract

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.

Keywords

Gene disruption Transposon Basidiomycetes Virulence factors 

References

  1. Adachi K, Nelson GH, Peoples KA, Frank SA, Montenegro-Chamorro MV, DeZwaan TM, Ramamurthy L, Shuster JR, Hamer L, Tanzer MM (2002) Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO. Curr Genet 42:123–127PubMedCrossRefGoogle Scholar
  2. Biery MC, Stewart FJ, Stellwagen AE, Raleigh EA, Craig NL (2000) A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis. Nucleic Acids Res 28:1067–1077PubMedCrossRefGoogle Scholar
  3. Buchanan K, Murphy JW (1998) What makes Cryptococcus neoformans a pathogen? Emerg Infect Dis 4:71–83PubMedGoogle Scholar
  4. Casadevall A, Perfect JR (1998) Cryptococcus neoformans. ASM Press, WashingtonGoogle Scholar
  5. Castano I, Kaur R, Pan S, Cregg R, De Las Penas A, Guo N, Biery MC, Craig NL, Cormack BP (2003) Tn7-based genome-wide random insertional mutagenesis in Candida glabrata. Genome Res 13:905–915PubMedCrossRefGoogle Scholar
  6. Chang YC, Kwon-Chung KJ (1994) Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14:4912–4919PubMedGoogle Scholar
  7. Chang YC, Kwon-Chung KJ (1998) Isolation of the third capsule-associated gene, CAP60, required for virulence in Cryptococcus neoformans. Infect Immun 66:2230–2236PubMedGoogle Scholar
  8. Chang YC, Kwon-Chung KJ (1999) Isolation, characterizatiom, and localization of a capsule-associated gene, CAP10, of Cryptococcus neoformans. J Bacteriol 181:5636–5643PubMedGoogle Scholar
  9. Chang YC, Penoyer L, Kwon-Chung KJ (1996) The second capsule gene of Cryptococcus neoformans CAP64 is essential for virulence. Infect Immun 64:1977–1983PubMedGoogle Scholar
  10. Cox GM, McDade HC, Chen SC, Tucker SC, Gottfredsson M, Wright LC, Sorrell TC, Leidich SD, Casadevall A, Ghannoum MA, Perfect JR (2001) Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. Mol Microbiol 39:166–175PubMedCrossRefGoogle Scholar
  11. Cox GM, Mukherjee J, Cole GT, Casadevall A, Perfect JR (2000) Urease as a virulence factor in experimental cryptococcosis. Infect Immun 68:443–448PubMedCrossRefGoogle Scholar
  12. Cox GM, Toffaletti DL, Perfect JR (1996) Dominant selection system for use in Cryptococcus neoformans. J Med Vet Mycol 34:385–391PubMedCrossRefGoogle Scholar
  13. Davidson RC, Cruz MC, Sia RA, Allen B, Alspaugh JA, Heitman J (2000) Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genet Biol 29:38–48PubMedCrossRefGoogle Scholar
  14. Davidson RC, Blankenship JR, Kraus PR, Berrios MDJ, Hull CM, D’Souza C, Wang P, Heitman J (2002) A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148:2607–2615PubMedGoogle Scholar
  15. Earley MC, Crouse GF (1998) The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 95:15487–15491PubMedCrossRefGoogle Scholar
  16. Edman JC, Keon-Chung KJ (1990) Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation. Mol Cell Biol 10:4538–4544PubMedGoogle Scholar
  17. Fox DS, Cruz MC, Sia RA, Ke H, Cox GM, Cardenas ME, Heitman J (2001) Calcinurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans. Mol Microbiol 39:835–849PubMedCrossRefGoogle Scholar
  18. Hamer L, Adachi K, Montenegro-Chamorro MV, Tanzer MM, Mahanty SK, Lo C, Tarpey RW, Skalchunes AR, Heiniger RW, Frank SA, Darveaux BA, Lampe DJ, Slater TM, Ramamurthy L, DeZwaan TM, Nelson GH, Shuster JR, Woessner J, Hamer JE (2001) Gene discovery and gene function assignment in filamentous fungi. Proc Natl Acad Sci USA 98:5110–5115PubMedCrossRefGoogle Scholar
  19. Hoang LM, Maguire JA, Doyle P, Fyfe M, Roscoe DL (2004) Cryptococcus neoformans infections at vancouver hospital and health sciences centre (1997–2002): Epidemiology, microbiology and histopathology. J Med Microbiol 53:935–940PubMedCrossRefGoogle Scholar
  20. Hua J, Meyer JD, Lodge JK (2000) Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans. Clin Diagn Lab Immunol 7:125–128PubMedGoogle Scholar
  21. Hull CM, Heitman J (2002) Genetics of Cryptococcus neoformans. Annu Rev Genet 36:557–615PubMedCrossRefGoogle Scholar
  22. Idnurm A, Reedy JL, Nussbaum JC, Heitman J (2004) Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell 3:420–429PubMedCrossRefGoogle Scholar
  23. Jadoun J, Shadkchan Y, Osherov N (2004) Disruption of the Aspergillus fumigatus argB gene using a novel in vitro transposon-based mutagenesis approach. Curr Genet 45:235–241PubMedCrossRefGoogle Scholar
  24. Kidd SE, Hagen F, Tscharke RL, Huynh M, Bartlett KH, Fyfe M, Macdougall L, Boekhout T, Kwon-Chung KJ, Meyer W (2004) A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci USA 101:17258–17263PubMedCrossRefGoogle Scholar
  25. Kidd SE, Guo H, Bartlett KH, Xu J, Kronstad JW (2005) Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas. Eukaryot Cell 10:1629–1638CrossRefGoogle Scholar
  26. Kwon -Chung KJ, Boekhout T, Fell JW, Diaz M (2002) Proposal to conserve the name Cryptococcus gatti against C. hondurianus and C. bacillisporus (Basidiomycoto, Hymenomycetes, Tremellomycetiadae) Taxon 51:804–806CrossRefGoogle Scholar
  27. Kwon-Chung KJ, Rhodes JC (1986) Ecapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans. Infect Immun 51:218–223PubMedGoogle Scholar
  28. Lodge JK, Jackson-Machelski E, Toffaletti DL, Perfect JR, Gordon JI (1994) Targeted gene replacement demonstrates that myristoyl-CoA: protein N-myristoyltransferase is essential for viability of Cryptococcus neoformans. Proc Natl Acad Sci USA 91:12008–12012PubMedCrossRefGoogle Scholar
  29. Loftus BJ, Fung E, Roncaglia P, Rowley D, Amedeo P, Bruno D, Vamathevan J, Miranda M, Anderson IJ, Fraser JA, Allen JE, Bosdet IE, Brent MR, Chiu R, Doering TL, Donlin MJ, D’Souza CA, Fox DS, Grinberg V, Fu J, Fukushima M, Haas BJ, Huang JC, Janbon G, Jones SJ, Koo HL, Krzywinski MI, Kwon-Chung JK, Lengeler KB, Maiti R, Marra MA, Marra RE, Mathewson CA, Mitchell TG, Pertea M, Riggs FR, Salzberg SL, Schein JE, Shvartsbeyn A, Shin H, Shumway M, Specht CA, Suh BB, Tenney A, Utterback TR, Wickes BL, Wortman JR, Wye NH, Kronstad JW, Lodge JK, Heitman J, Davis RW, Fraser CM, Hyman RW (2005) The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans Science 307:1321–1324PubMedCrossRefGoogle Scholar
  30. Moyrand F, Klaproth B, Himmerlreich U, Dromer F, Janbon G (2002) Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans. Mol Microbiol 45:837–849PubMedCrossRefGoogle Scholar
  31. Nelson RT, Pryor BA, Lodge JK (2003) Sequence length required for homologous recombination in Cryptococcus neoformans. Fungal Genet Biol 38:1–9PubMedCrossRefGoogle Scholar
  32. Lian TS, Simmer MI, D’Souza CA, Steen BR, Zuyderduyn SD, Jone SJM, Marra MA, Kronstad JW (2004) Iron-regulated transcription and capsule formation in the fungal pathogen Cryptococcus neoformans. Mol Microbiol 55:1452–1472CrossRefGoogle Scholar
  33. Perfect JR (2005) Cryptococcus neoformans: a sugar-coated killer with designer genes. FEMS Immunol Med Microbiol 45:395–404PubMedCrossRefGoogle Scholar
  34. 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 142:1557–1565PubMedCrossRefGoogle Scholar
  35. Rayssiguier C, Thaler DS, Radma (1989) The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 23:396–401CrossRefGoogle Scholar
  36. Salas SD, Bennet JE, Kwon-Chung KJ, Perfect JR, Williamson PR (1996) Effect of the laccase gene, CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med 184:377–386PubMedCrossRefGoogle Scholar
  37. Sia RA, Lengeler KB, Heitman J (2000) Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Fungal Genet Biol 29:153–163PubMedCrossRefGoogle Scholar
  38. Solomon PS, Lee RC, Greer Wilson TJ, Oliver RP (2004) Pathogenicity of Stagonospora nodorum requires malate synthase. Mol Microbiol 53:1065–1073PubMedCrossRefGoogle Scholar
  39. Steen BR, Lian T, Zuyderduyn S, MacDonald WK, Marra M, Jones SJ, Kronstad JW (2002) Temperature-regulated transcription in the pathogenic fungus Cryptococcus neoformans. Genome Res 12:1386–1400PubMedCrossRefGoogle Scholar
  40. Steen BR, Zuyderduyn S, Toffaletti DL, Marra M, Jones SJ, Perfect JR, Kronstad JW (2003) Cryptococcus neoformans gene expression during experimental cryptococcal meningitis. Eukaryot Cell 2:1336–1349PubMedCrossRefGoogle Scholar
  41. Zwiers L, De Waard MA (2001) Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphaerella graminicola. Curr Genet 39:388–393PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.The Michael Smith LaboratoriesThe University of British ColumbiaVancouverCanada

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