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Volume 160, Issue 10

Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Free

Abstract

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte (anamorph: ), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the gene adapted to the pathogenic yeast () or a synthetic codon-optimized () gene with neomycin resistance () cassette for one-step marker excision. Both genes were under control of the copper-repressible promoter (). Molecular analyses of resultant transformants showed that only the -harbouring module was functional in . Applying this system, we successfully produced the recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) () or serine protease () genes, producing mutant strains with double or triple gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

  • Received:
  • Accepted:
  • Published Online:
Funding
This study was supported by the:
  • Ministry of Education, Culture, Sports, Science and Technology of Japan (Award 23590520)
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2014-10-01
2024-04-19
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References

  1. Alshahni M. M., Makimura K., Yamada T., Satoh K., Ishihara Y., Takatori K., Sawada T. ( 2009). Direct colony PCR of several medically important fungi using Ampdirect plus. Jpn J Infect Dis 62:164–167[PubMed]
     [Google Scholar]
  2. Alshahni M. M., Makimura K., Yamada T., Takatori K., Sawada T. ( 2010). Nourseothricin acetyltransferase: a new dominant selectable marker for the dermatophyte Trichophyton mentagrophytes.. Med Mycol 48:665–668 [View Article][PubMed]
     [Google Scholar]
  3. Alshahni M. M., Yamada T., Takatori K., Sawada T., Makimura K. ( 2011). Insights into a nonhomologous integration pathway in the dermatophyte Trichophyton mentagrophytes: efficient targeted gene disruption by use of mutants lacking ligase IV. Microbiol Immunol 55:34–43 [View Article][PubMed]
     [Google Scholar]
  4. Belteki G., Gertsenstein M., Ow D. W., Nagy A. ( 2003). Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase. Nat Biotechnol 21:321–324 [View Article][PubMed]
     [Google Scholar]
  5. Broach J. R., Guarascio V. R., Jayaram M. ( 1982). Recombination within the yeast plasmid 2μm circle is site-specific. Cell 29:227–234 [View Article][PubMed]
     [Google Scholar]
  6. Caponigro G., Muhlrad D., Parker R. ( 1993). A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons. Mol Cell Biol 13:5141–5148[PubMed]
     [Google Scholar]
  7. Cardoza R. E., Gutiérrez S., Ortega N., Colina A., Casqueiro J., Martín J. F. ( 2003). Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH-regulated promoters and secretion using two different pre-pro sequences. Biotechnol Bioeng 83:249–259 [View Article][PubMed]
     [Google Scholar]
  8. Critchlow S. E., Jackson S. P. ( 1998). DNA end-joining: from yeast to man. Trends Biochem Sci 23:394–398 [View Article][PubMed]
     [Google Scholar]
  9. Dymecki S. M. ( 1996). Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc Natl Acad Sci U S A 93:6191–6196 [View Article][PubMed]
     [Google Scholar]
  10. Giddey K., Favre B., Quadroni M., Monod M. ( 2007a). Closely related dermatophyte species produce different patterns of secreted proteins. FEMS Microbiol Lett 267:95–101 [View Article][PubMed]
     [Google Scholar]
  11. Giddey K., Monod M., Barblan J., Potts A., Waridel P., Zaugg C., Quadroni M. ( 2007b). Comprehensive analysis of proteins secreted by Trichophyton rubrum and Trichophyton violaceum under in vitro conditions. J Proteome Res 6:3081–3092 [View Article][PubMed]
     [Google Scholar]
  12. Girardin H., Latge J. P. ( 1994). DNA extraction and quantification. Molecular Biology of Pathogenic Fungi5–9 Maresca B., Kobayashi G. S. New York: Telos Press;
     [Google Scholar]
  13. Gonzalez R., Ferrer S., Buesa J., Ramon D. ( 1989). Transformation of the dermatophyte Trichophyton mentagrophytes to hygromycin B resistance. Infect Immun 57:2923–2925[PubMed]
     [Google Scholar]
  14. Gooch V. D., Mehra A., Larrondo L. F., Fox J., Touroutoutoudis M., Loros J. J., Dunlap J. C. ( 2008). Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock. Eukaryot Cell 7:28–37 [View Article][PubMed]
     [Google Scholar]
  15. Gouka R. J., Punt P. J., van den Hondel C. A. M. J. J. ( 1997). Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels. Appl Environ Microbiol 63:488–497[PubMed]
     [Google Scholar]
  16. Grumbt M., Defaweux V., Mignon B., Monod M., Burmester A., Wöstemeyer J., Staib P. ( 2011a). Targeted gene deletion and in vivo analysis of putative virulence gene function in the pathogenic dermatophyte Arthroderma benhamiae.. Eukaryot Cell 10:842–853 [View Article][PubMed]
     [Google Scholar]
  17. Grumbt M., Monod M., Staib P. ( 2011b). Genetic advances in dermatophytes. FEMS Microbiol Lett 320:79–86 [View Article][PubMed]
     [Google Scholar]
  18. Gustafsson C., Govindarajan S., Minshull J. ( 2004). Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353 [View Article][PubMed]
     [Google Scholar]
  19. Idnurm A., Howlett B. J. ( 2003). Analysis of loss of pathogenicity mutants reveals that repeat-induced point mutations can occur in the Dothideomycete Leptosphaeria maculans.. Fungal Genet Biol 39:31–37 [View Article][PubMed]
     [Google Scholar]
  20. Iwata A., Alshahni M. M., Nishiyama Y., Makimura K., Abe S., Yamada T. ( 2012). Development of a tightly regulatable copper-mediated gene switch system in dermatophytes. Appl Environ Microbiol 78:5204–5211 [View Article][PubMed]
     [Google Scholar]
  21. Jacobson A., Peltz S. W. ( 1996). Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem 65:693–739 [View Article][PubMed]
     [Google Scholar]
  22. Jousson O., Léchenne B., Bontems O., Capoccia S., Mignon B., Barblan J., Quadroni M., Monod M. ( 2004a). Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum.. Microbiology 150:301–310 [View Article][PubMed]
     [Google Scholar]
  23. Jousson O., Léchenne B., Bontems O., Mignon B., Reichard U., Barblan J., Quadroni M., Monod M. ( 2004b). Secreted subtilisin gene family in Trichophyton rubrum.. Gene 339:79–88 [View Article][PubMed]
     [Google Scholar]
  24. Koda A., Bogaki T., Minetoki T., Hirotsune M. ( 2005). High expression of a synthetic gene encoding potato α-glucan phosphorylase in Aspergillus niger.. J Biosci Bioeng 100:531–537 [View Article][PubMed]
     [Google Scholar]
  25. Kopke K., Hoff B., Kück U. ( 2010). Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes. Appl Environ Microbiol 76:4664–4674 [View Article][PubMed]
     [Google Scholar]
  26. Kopke K., Hoff B., Bloemendal S., Katschorowski A., Kamerewerd J., Kück U. ( 2013). Members of the Penicillium chrysogenum velvet complex play functionally opposing roles in the regulation of penicillin biosynthesis and conidiation. Eukaryot Cell 12:299–310 [View Article][PubMed]
     [Google Scholar]
  27. Martinez D. A., Oliver B. G., Gräser Y., Goldberg J. M., Li W., Martinez-Rossi N. M., Monod M., Shelest E., Barton R. C. & other authors ( 2012). Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection. MBio 3:e00259–e12 [View Article][PubMed]
     [Google Scholar]
  28. Monod M., Léchenne B., Jousson O., Grand D., Zaugg C., Stöcklin R., Grouzmann E. ( 2005). Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum.. Microbiology 151:145–155 [View Article][PubMed]
     [Google Scholar]
  29. Morschhäuser J., Michel S., Staib P. ( 1999). Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination. Mol Microbiol 32:547–556 [View Article][PubMed]
     [Google Scholar]
  30. Nelson G., Kozlova-Zwinderman O., Collis A. J., Knight M. R., Fincham J. R., Stanger C. P., Renwick A., Hessing J. G., Punt P. J. & other authors ( 2004). Calcium measurement in living filamentous fungi expressing codon-optimized aequorin. Mol Microbiol 52:1437–1450 [View Article][PubMed]
     [Google Scholar]
  31. Rawlings N. D., Barrett A. J., Bateman A. ( 2012). MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 40:D1D343–D350 [View Article][PubMed]
     [Google Scholar]
  32. Raymond C. S., Soriano P. ( 2007). High-efficiency FLP and ΦC31 site-specific recombination in mammalian cells. PLoS ONE 2:e162 [View Article][PubMed]
     [Google Scholar]
  33. Reuss O., Vik A., Kolter R., Morschhäuser J. ( 2004). The SAT1 flipper, an optimized tool for gene disruption in Candida albicans.. Gene 341:119–127 [View Article][PubMed]
     [Google Scholar]
  34. Sadowski P. D. ( 1995). The Flp recombinase of the 2-µm plasmid of Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol 51:53–91 [View Article][PubMed]
     [Google Scholar]
  35. Schweizer H. P. ( 2003). Applications of the Saccharomyces cerevisiae Flp-FRT system in bacterial genetics. J Mol Microbiol Biotechnol 5:67–77 [View Article][PubMed]
     [Google Scholar]
  36. Song H., Niederweis M. ( 2007). Functional expression of the Flp recombinase in Mycobacterium bovis BCG. Gene 399:112–119 [View Article][PubMed]
     [Google Scholar]
  37. Sriranganadane D., Waridel P., Salamin K., Feuermann M., Mignon B., Staib P., Neuhaus J. M., Quadroni M., Monod M. ( 2011). Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH. Proteomics 11:4422–4433 [View Article][PubMed]
     [Google Scholar]
  38. Staib P., Kretschmar M., Nichterlein T., Köhler G., Michel S., Hof H., Hacker J., Morschhäuser J. ( 1999). Host-induced, stage-specific virulence gene activation in Candida albicans during infection. Mol Microbiol 32:533–546 [View Article][PubMed]
     [Google Scholar]
  39. Staib P., Zaugg C., Mignon B., Weber J., Grumbt M., Pradervand S., Harshman K., Monod M. ( 2010). Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection. Microbiology 156:884–895 [View Article][PubMed]
     [Google Scholar]
  40. Sternberg N., Hamilton D. ( 1981). Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol 150:467–486 [View Article][PubMed]
     [Google Scholar]
  41. Tan X., Liang F., Cai K., Lu X. ( 2013). Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants. Appl Microbiol Biotechnol 97:6373–6382 [View Article][PubMed]
     [Google Scholar]
  42. Te’o V. S. J., Cziferszky A. E., Bergquist P. L., Nevalainen K. M. ( 2000). Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei. FEMS Microbiol Lett 190:13–19 [View Article][PubMed]
     [Google Scholar]
  43. Tokuoka M., Tanaka M., Ono K., Takagi S., Shintani T., Gomi K. ( 2008). Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression. Appl Environ Microbiol 74:6538–6546 [View Article][PubMed]
     [Google Scholar]
  44. Uchida K., Tanaka T., Yamaguchi H. ( 2003). Achievement of complete mycological cure by topical antifungal agent NND-502 in guinea pig model of tinea pedis. Microbiol Immunol 47:143–146 [View Article][PubMed]
     [Google Scholar]
  45. Ueno K., Uno J., Nakayama H., Sasamoto K., Mikami Y., Chibana H. ( 2007). Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata.. Eukaryot Cell 6:1239–1247 [View Article][PubMed]
     [Google Scholar]
  46. Vogt K., Bhabhra R., Rhodes J. C., Askew D. S. ( 2005). Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus.. BMC Microbiol 5:1 [View Article][PubMed]
     [Google Scholar]
  47. Yamada T., Makimura K., Uchida K., Yamaguchi H. ( 2005). Reproducible genetic transformation system for two dermatophytes, Microsporum canis and Trichophyton mentagrophytes.. Med Mycol 43:533–544 [View Article][PubMed]
     [Google Scholar]
  48. Yamada T., Makimura K., Hisajima T., Ito M., Umeda Y., Abe S. ( 2008). Genetic transformation of the dermatophyte, Trichophyton mentagrophytes, based on the use of G418 resistance as a dominant selectable marker. J Dermatol Sci 49:53–61 [View Article][PubMed]
     [Google Scholar]
  49. Yamada T., Makimura K., Satoh K., Umeda Y., Ishihara Y., Abe S. ( 2009a). Agrobacterium tumefaciens-mediated transformation of the dermatophyte, Trichophyton mentagrophytes: an efficient tool for gene transfer. Med Mycol 47:485–494 [View Article][PubMed]
     [Google Scholar]
  50. Yamada T., Makimura K., Hisajima T., Ishihara Y., Umeda Y., Abe S. ( 2009b). Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes.. FEMS Microbiol Lett 298:208–217 [View Article][PubMed]
     [Google Scholar]
  51. Zaugg C., Monod M., Weber J., Harshman K., Pradervand S., Thomas J., Bueno M., Giddey K., Staib P. ( 2009). Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins. Eukaryot Cell 8:241–250 [View Article][PubMed]
     [Google Scholar]
  52. Zhang A., Lu P., Dahl-Roshak A. M., Paress P. S., Kennedy S., Tkacz J. S., An Z. ( 2003). Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis.. Mol Genet Genomics 268:645–655[PubMed]
     [Google Scholar]
  53. Zhang X., Wang Y., Chi W., Shi Y., Chen S., Lin D., Jin Y. ( 2014). Metalloprotease genes of Trichophyton mentagrophytes are important for pathogenicity. Med Mycol 52:36–45[PubMed]
     [Google Scholar]
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Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii
Microbiology 160, 2122 (2014); https://doi.org/10.1099/mic.0.076562-0
/content/journal/micro/10.1099/mic.0.076562-0
/content/journal/micro/10.1099/mic.0.076562-0
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