Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans

Nature Biotechnology volume 19, pages 235–241 (2001)Cite this article

A Corrigendum to this article was published on 01 July 2001

Abstract

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Integrative C. albicans vectors.
Figure 2: Integration of library plasmids into the C. albicans genome.
Figure 3: Growth curves of 74 transformants (wells 1–74 and 101–174, respectively) and from CAI-4 (wells 75 and 175, respectively) grown for three days in antisense (A) noninducing (S medium pH 6, containing 50 mM lithium acetate and 2% glucose; wells 1–74) and (B) inducing (S medium pH 6, containing 50 mM lithium acetate and 2% galactose; wells 101–174) conditions in Honeywell 100-well plates (Labsystems).
Figure 4: Activity of the C. albicans GAL1 promoter in the absence and presence of maltose as a carbon source.
Figure 5: Expression analysis of genes targeted in clones 36 and 38.

Similar content being viewed by others

References

  1. Fox, J.L. Fungal infection rates are increasing. ASM News 10, 515–518 (1993).

    Google Scholar 

  2. Fisher Hoch, S.P. & Hutwagner, L. Opportunistic candidiasis: an epidemic of the 1980s. Clin. Infect. Dis. 21, 897–904 (1995).

    Article  CAS  Google Scholar 

  3. Groll, A.H., Piscitelli, S.C. & Walsh, T.J. Clinical pharmacology of systemic antifungal agents: a comprehensive review of agents in clinical use, current investigational compounds, and putative targets for antifungal drug development. Adv. Pharmacol. 44, 343–501 (1998).

    Article  CAS  Google Scholar 

  4. Jack, D.B. The search for new antifungal drugs and strategies continues. Drug News Perspect. 11, 306–309 (1998).

    CAS  PubMed  Google Scholar 

  5. Smith, V., Botstein, D. & Brown, P.O. Genetic footprinting: a genomic strategy for determining a gene's function given its sequence. Proc. Natl. Acad. Sci. USA 92, 6479–6483 (1995).

    Article  CAS  Google Scholar 

  6. Fairhead, C., Thierry, A., Denis, F., Eck, M. & Dujon, B. “Mass-murder” of ORFs from three regions of chromosome XI from S. cerevisiae. Gene 223, 33–46 (1998).

    Article  CAS  Google Scholar 

  7. Magee, B.B. & Magee, P.T. Induction of mating in Candida albicans by construction of MTLa and MTLα Strains. Science 14, 310–313 (2000).

  8. Hull, C.M., Raisner, R.M. & Johnson, A.D. Evidence for mating of the “asexual” yeast Candida albicans in a mammalian host. Science 14, 307–310 (2000).

  9. Matthews, G.D., Goodwin, T.J., Butler, M.I., Berryman, T.A. & Poulter, R.T. pCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans. J. Bacteriol. 179, 7118–7128 (1997).

    Article  CAS  Google Scholar 

  10. Pla, J., Gil, C., Monteoliva, F., Sanchez, M. & Nombela, C. Understanding Candida albicans at the molecular level. Yeast 12, 1677–1702 (1996).

    Article  CAS  Google Scholar 

  11. Fonzi, W.A. & Irwin, M.Y. Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Wilson, R.B., Davis, D. & Mitchell, A.P. Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J. Bacteriol. 181, 1868–1874 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. de Hoogt, R., Contreras, R., Luyten, W.H.M.L. & De Backer, M.D. PCR- and ligation-mediated synthesis of split-marker cassettes with long flanking homology regions for gene disruption in Candida albicans. BioTechniques 28, 1112–1116 (2000).

    Article  CAS  Google Scholar 

  14. De Backer, M.D., Pla J. & Magee P. Recent developments in molecular genetics of Candida albicans [review]. Annu. Rev. Microbiol. 54, 463–498 (2000).

    Article  CAS  Google Scholar 

  15. Del Rosario, M., Stephans, J.C., Zakel, J., Escobedo, J. & Giese, K. Positive selection system to screen for inhibitors of human immunodeficiency virus-1 transcription. Nat. Biotechnol. 14, 1592–1596 (1996).

    Article  CAS  Google Scholar 

  16. Atkins, D., Arndt, G.M. & Izant, J.G. . Antisense gene expression in yeast. Biol. Chem. Hoppe-Seyler 375, 721–729 (1994).

    CAS  PubMed  Google Scholar 

  17. Atkins, D. & Gerlach, W.L. Artificial ribozyme and antisense gene expression in S. cerevisiae. Antisense Res. Dev. 4, 109–117 (1994).

    Article  CAS  Google Scholar 

  18. Giese, K. Method and construct for screening for inhibitors of transcriptional activation. International patent application WO 97/10360 (1997).

  19. De Backer, M.D. et al. Transformation of Candida albicans by electroporation. Yeast 15, 1609–1618 (1999).

    Article  CAS  Google Scholar 

  20. Ferbeyre, G., Bratty, J., Chen, H. & Cedergren, R. . Cell cycle arrest promotes trans-hammerhead ribozyme action in yeast. J. Biol. Chem. 271, 19318–19323 (1996).

    Article  CAS  Google Scholar 

  21. Nelissen, B., Mordant, P., Jonniaux, J-L., De Wachter, R. & Goffeau, A. Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing. FEBS Lett. 377, 232–236 (1996).

    Article  Google Scholar 

  22. Fraser, C.M. et al. The minimal gene complement of Mycoplasma genitalium. Science 270, 397–403 (1995).

    Article  CAS  Google Scholar 

  23. Fleischmann, R.D. et al. Whole-genome random sequencing and assembly of Haemophilus influenzae. Science 269, 496–512 (1995).

    Article  CAS  Google Scholar 

  24. Sinclair, D.A. & Guarente, L. Extrachromosomal rDNA circles—a cause of aging in yeast. Cell 91, 1033–1042 (1997).

    Article  CAS  Google Scholar 

  25. Reeder, R.H. & Lang, W.H. Terminating transcription in eukaryotes: lessons learned from RNA polymerase I. Trends Biochem. Sci. 22, 473–477 (1997).

    Article  CAS  Google Scholar 

  26. Moehle, C.M. Patent application RiboGene Inc., PCT WO 95/11969 (1995).

  27. Numata, K., Yamamoto, H., Hatori, M., Miyaki, T. & Kawaguchi, H. Isolation of an aminoglycoside hypersensitive mutant and its application in screening. J. Antibiot. 39, 994–1000 (1986).

    Article  CAS  Google Scholar 

  28. Oliver, S. Redundancy reveals drugs in action. Nat. Genet. 21, 245–246 (1999).

    Article  CAS  Google Scholar 

  29. Jensen-Pergakes, K.L. et al. Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants. Antimicrob. Agents. Chemother. 42, 1160–1167 (1998).

    Article  CAS  Google Scholar 

  30. De Backer, M.D. et al. Single allele knockout of C. albicans CGT1 leads to unexpected resistance to hygromycin B and elevated temperature. Microbiology 146, 353–365 (2000).

    Article  CAS  Google Scholar 

  31. Blin, N. & Stafford, D.W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303–2308 (1976).

    Article  CAS  Google Scholar 

  32. Zhu, J., Kempenaers, W., Van der Straeten, D., Contreras, R. & Fiers, W. A method for fast and pure DNA elution from agarose gels by centrifugal filtration. Bio/Technology 3, 1014–1016 (1985).

    CAS  Google Scholar 

  33. Leuker, C.E., Sonneborn, A., Delbruck, S. & Ernst, J.F. Sequence and promoter regulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans. Gene 192, 235–240 (1997).

    Article  CAS  Google Scholar 

  34. Orlando, C., Pinzani, P. & Pazzagli, M. Developments in quantitative PCR. Clin. Chem. Lab. Med. 36, 255–269 (1998).

    Article  CAS  Google Scholar 

  35. Lie, Y.S. & Petropoulos, C.J. Advances in quantitative PCR technology, 5′ nuclease assays. Curr. Opin. Biotechnol. 9, 43–48 (1998).

    Article  CAS  Google Scholar 

  36. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual, Edn. 2. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1989).

    Google Scholar 

  37. De Backer, M.D. et al. Genomic cloning, heterologous expression and pharmacological characterization of a human histamine H1 receptor. Biochem. Biophys. Res. Commun. 197, 1601–1608 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. J. Vialard, Dr. G. Froyen, Dr. T. Ilyina, H. Vanden Bossche, T. Thielemans, Dr. R. Gordon, N. Delcroix, S. De Breucker, K. De Wagter, A. Diels, M. Hijzen, G. Verheyen, and I. Van de Weyer (all at Janssen Pharmaceutica Beerse) for much appreciated contributions. We are grateful to J. Kaufman (R.W. Johnson Pharmaceutical Research Institute, San Diego, CA) for graphical support. This work was supported in Belgium by the IWT (grant 960192) and VIB.

Author information

Authors and Affiliations

  1. Department of Advanced Bio-Technologies, Janssen Pharmaceutica, Turnhoutseweg 30, Beerse, B2340, Belgium

    Marianne D. De Backer, Bart Nelissen, Inge Loonen, Sandy Vandoninck, Ronald de Hoogt, Fermin A. Simons & Walter H.M.L. Luyten

  2. Department of Biotechnology and High Throughput Screening, Janssen Pharmaceutica, Turnhoutseweg 30, Beerse, B2340, Belgium

    Peter Verhasselt & Greet Vanhoof

  3. Department of Molecular Biology, Subunit Fundamental and Applied Molecular Biology, University Gent and Vlaams Instituut voor de bevordering van het Wetenschappelijk Technologisch onderzoek in de industrie (IWT/VIB), K.L. Ledeganckstraat 35, Gent, B9000, Belgium

    Marc Logghe, Jasmine Viaene, Sylviane Dewaele & Roland Contreras

Authors
  1. Marianne D. De Backer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bart Nelissen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marc Logghe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jasmine Viaene
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Inge Loonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sandy Vandoninck
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ronald de Hoogt
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sylviane Dewaele
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Fermin A. Simons
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Peter Verhasselt
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Greet Vanhoof
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Roland Contreras
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Walter H.M.L. Luyten
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marianne D. De Backer.

Rights and permissions

Reprints and permissions

About this article

Cite this article

De Backer, M., Nelissen, B., Logghe, M. et al. An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans. Nat Biotechnol 19, 235–241 (2001). https://doi.org/10.1038/85677

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/85677

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing