Candida albicans and the ICL1 gene

1. Candida albicans and the ICL1 gene constructing a double knock-out Matthijs Dekkers Supervisor: Els Mol Mei/Juni 2005

2. Introduction • Candida albicans, a fungal pathogen that causes opportunistic infections • Systemic fungal infections in immunocompromised • Increased prevalence and severity with rise of AIDS, chemotherapy, organ transplantation

3. Introduction • Candida is the focus of many studies • Understanding pathogenesis • Finding new targets for antifungals

4. Dimorphism • Ability to switch between yeast and hyphal growth • Essential for its pathogenicity • Allows escape from macrophage

5. Phagocytosis • C. albicans is phagocytosed by macrophages and neutrophils • Normally destroyed in the phagolysosome • C. albicans can use different carbon sources, by rapidly adapting its metabolism • This allows C. albicans to grow hyphae and perforate the plasma membrane of the macrophage • Releases the fungal cell while killing the macrophage

6. Glyoxylate Cycle • Glucose is the preferred carbon source in most organisms • Glyoxylate cycle allows use of C2 carbon compounds • Studies have shown that genes are upregulated after phagocytosis

7. Isocitrate Lyase • Isocitrate lyase, product of the CaICL1 gene, is a key enzyme • Hydrolyzes isocitrate (C6) to succinate (C4) • Activity both specific and limited to glyoxylate cycle • ICL1 shares homology with other fungi, plants, bacteria but not mammals • This makes it a possible target for antifungal agents

8. Aim of this Study To allow investigation of the role of isocitrate lyase in the pathogenicity of C. albicans, in this study deletion mutants for the ICL1 gene will be constructed. PCR-based gene targeting involving homologous recombination will be used to create a double knock-out strain of C. albicans.

9. Materials & Methods

10. ICL1 ARG4 PCR-based Gene Manipulation • PCR amplification of cassettes containing markers (ARG4 or HIS1) • Primers have 80 bases corresponding with ICL1 at its 5’ end 20 bases with the 3’ end of the cassette • PCR results in a construct with a marker gene and 80bp homology with ICL1 on both ends • A second PCR with 38b overlapping primers ensures homology ARG4 ARG4

11. PCR-based Gene Manipulation • When introduced to Candida so that homologous recombination occurs, the marker gene replaces the target gene (ICL1), resulting in a single knock-out • A second knock-out will be done in the same way, using a different marker

12. PCR ARG4 PCR-based Gene Manipulation ICL1 ARG4 ARG4 recombination

13. BWP-17 • BWP17 strain bears 3 deletions in auxotrophic genes: URA3, HIS1, ARG4 allowing selection for transformants on plates lacking histidine or arginine

14. Verification of Transformants • PCR technique will be used to verify the correct integration of the marker and thus the knock-out • different combinations of marker specific and ICL1 specific primers will give definitive answer whether transformants are ICL1 knock-outs

15. FP A4 PCR A5 RP ARG4 Verification of Transformants FP+A4 A5+RP FP+RP

16. Results

17. Cassette amplification • Amplification of ARG4 and HIS1 Wendland cassettes with ICL1 homologous primers results in 1900bp and 1428bp fragments

18. Cassette amplification • Amplification of ARG4 and HIS1 Wendland cassettes with ICL1 homologous primers results in 1900bp and 1428bp fragments • PCR products will be elongated and used in transformation HIS1 ±1400bp ARG4 ±1900bp

19. First Transformation • 2 transformations, 1 with HIS1, 1 with ARG4 were done on BWP17 and plated on resp. SD -his and SD -arg plates • resulting transformants were analyzed with PCR

20. First Transformation FP A4 ARG4 A5 RP PCR +ARG4 +ARG4 +HIS1 +HIS1 BWP17

21. First Transformation FP A4 ARG4 A5 RP PCR +ARG4 +ARG4 +HIS1 +HIS1 BWP17 1536bp

22. First Transformation FP A4 ARG4 A5 RP PCR +ARG4 +ARG4 +HIS1 +HIS1 BWP17 789bp

23. First Transformation FP A4 ARG4 A5 RP PCR +ARG4 +ARG4 +HIS1 +HIS1 BWP17 ARG4: 2369 ICL1: 2152

24. CMD1 CMD2 icl1Δ::ARG4 /ICL1 First Transformation • CMD1 and CMD2 were both verified by PCR to be icl1Δ::ARG4 /ICL1 • These will be used for further experiments in this study

25. Second Transformation • The second transformation proved to be harder than the first • Histidine cassette integrated in genome, but not in ICL1 locus • A new screening technique was used

26. A Different Approach • Transformants lacking both ICL1 alleles should show attenuated growth on ethanol/acetate plates • All double transformants were plated on: • YPD • YNB + 2% glucose • YNB + 2% acetate • YNB + 2% ethanol (+uri, +his, +arg)

27. A Double Knock-Out? • 1 transformant did not grow at all, both on acetate and ethanol plates, but showed normal growth on YPD and YNB + glucose. This strain was further analyzed YNB + 2% acetate YPD

28. Second Transformation FP A4 ARG4 A5 RP PCR CMD1 CMD3 BWP17 1536bp

29. Second Transformation FP A4 ARG4 A5 RP PCR CMD1 CMD3 BWP17 789bp

30. H5 H4 RP Second Transformation FP HIS1 HIS1 PCR CMD1 CMD3 BWP17 415bp

31. H5 H4 RP Second Transformation FP HIS1 HIS1 PCR 400bp CMD1 CMD3 BWP17

32. RP Second Transformation FP HIS1 FP ARG4 PCR RP 2369bp CMD1 1781bp CMD3 BWP17

33. A Double Knock-Out • CMD3 was verified by selection on –arg, -his plates and by PCR to be icl1Δ::ARG4 /icl1Δ::HIS1 CMD3 icl1Δ::ARG4 /icl1Δ::HIS1

34. Future Research • Complementation of CMD3 with ICL1 • Growth comparison on different media • wild type • BWP17 • CMD1, CMD2 and CMD3 • complemented CMD3 • Virulence tests in mice

35. Acknowledgements thanks to everybody, especially Els Mol

36. Questions & Discussion