1. Costi Sifri and Brian Enloe Ausubel Group Meeting April 1, 2003 Candida - C. elegans Model System

2. Candida and Human Disease C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. dubliniensis, and hundreds more commensal organisms of the mucous membranes the most common human fungal pathogens opportunistic pathogens of the mucous membranes and skin, bloodstream, urinary tract, and deep organs 4th most common cause of nosocomial bloodstream infections (after coagulase-negative staphylococci, Staphylococcus aureus, and enterococci) with an attributable mortality of 38%* increasing drug resistance (emergence of non-albicans Candida) Wey SB, et al. Arch. Intern. Med. 148:2349-53, 1988. Edmond MB, et al. Clin. Infect. Dis. 29:239-44, 1999.

3. Clinical Candida isolates kill C. elegans on modified NGM at 25 o C

4. Survival (percent) Time (hours) 020406080 0 25 50 75 100 C. albicans CAN14 - alive CAN14 - amphoB killed C. glabrata BG2 - alive BG2 - amphoB killed C. elegans killing by Candida requires live yeast

5. Candida accumulates within the C. elegans intestine C. albicans/GFP - 20 hour incubation Anterior Posterior

6. Survival (percent) Time (hours) 0204060 0 25 50 75 100 CAN14 CAN14-OP50 6h CAN14-OP50 18h CAN14-OP50 22h C. elegans can be rescued from C. albicans by transfer to an innocuous food source

7. Survival (percent) Time (hours) 0100200300 0 25 50 75 100 Ca : N2 Ca : glp1 Matricide is a significant component of C. elegans killing by C. albicans...and may be the sole mechanism of killing by S. cerevisiae Sc : N2 Sc : glp1

8. Rapid killing (LT 50  28-32 hours) by many Candida clinical isolates and lab strains Requires live yeast Yeast colonize the nematode alimentary tract Colonization is not persistent Substantial matricide (severe bagging) Media dependant : modified NGM > TSA > BHI Temperature dependant All stages are killed : Young Adults > L4s > L1s Young larvae do not grow or mature on Candida Characteristics of a C. elegans - Candida Model System

9. Candida accumulates within the L1 intestine but does not support worm maturation and growth L1 larvae on C. glabrata - 48 hour incubation

10. Candida Virulence Factors Host Recognition and Adherence –adhesins, biofilm Morphogenesis - Filamentation –transition between unicellular yeast and filamentous growth Secreted Enzymes –secreted aspartic proteinases (SAPs), phospholipases Phenotypic Switching –white-opaque, fuzzy-smooth, smooth-star Metabolic Enzymes –URA3, ADE2, HIS1, FAS2

11. Invasion of esophageal tissue with C. albicans

12. Candida albicans Filamentation Pathway Starvation Ras1 MAP kinase cascade Cph1 CELL MORPHOLOGY Cyr1 cAMP Efg1 ? ? GENE EXPRESSION Tup1

13. CAN14 wild-type CAN34 efg1/efg1 cph1/cph1 Morphology of C. albicans wildtype and efg1-cph1 mutant

14. The Efg1 and Cph1 filamentation signaling pathways are important for virulence in mice 10 5 10 7 10 5 10 7 Lo HJ, et al. Cell 90:939-49, 1997

15. Survival (percent) Time (survival) 050100 0 25 50 75 100 The Efg1 and Cph1 filamentation signaling pathways are important in C. elegans killing Cph1 = MAP kinase pathway Efg1 = cAMP PKA pathway wild-type cph1 efg1 efg1 cph1 efg1 / EFG1 efg1 cph1 / EFG1 COMPLEMENTS

16. CAN14 (wt) CAN34 (efg1/efg1 cph1/cph1) Anterior C. albicans does not filament within the nematode intestine

17. CAN14 (wt) CAN34 (efg1/efg1 cph1/cph1) Posterior C. albicans does not filament within the nematode intestine

18. CAN14 (wt) CAN34 (efg1/efg1 cph1/cph1) Close-up

19. Survival (percent) Time (hours) 0204060 0 25 50 75 100 Wild type Ca  sap1-3  sap4-6 Secreted Aspartic Proteinase (SAP) mutants are not attenuated in Candida-mediated C. elegans killing Efg1 regulates SAP4, SAP5, and SAP6 gene expression

20. Time (hours) 020406080 0 25 50 75 100 Survival (percent) CAN14 (wildtype) CAN14 : pepA CAN34 (efg1 cph1) Inhibition of SAPs with Pepstatin A does not alter Candida-mediated C. elegans killing

21. Candida albicans Filamentation Pathway Starvation Ras1 MAP kinase cascade Cph1 CELL MORPHOLOGY Cyr1 cAMP Efg1 ? ? GENE EXPRESSION Tup1

22. 0204060 0 25 50 75 100 Time (survival) Survival (percent) wild-type ras1 tup1 Ras1 and Tup1 are not important in C. elegans killing

23. Nematodes are able to consume filamentous Candida CAN39 (tup1/tup1)

24. Screen: Construction of a Candida mutant library Species: C. glabrata (haploid) Strain: BG14 (ura3- auxotroph of BG2) Vector: YIplac211 Strategy: Random mutagenesis via nonhomologous recombination after transformation with linearized YIplac211 Selection: Ura3+ Transformation efficiency: ~ 100/  g Cormack BP, et al. Science 285:578-82, 1999. Cormack BP, el al. Genetics 151:979-87, 1999.

25. Primary 1028 mutants Candida glabrata screen Secondary 74 mutants Tertiary 18 of 71 are attenuated 3 left to test Auxotrophic on NGM 14 mutants 12 are attenuated

26. Time (hours) 0204060 0 25 50 75 100 Survival (percent) BG2 ace189 A representative C. glabrata mutant (ace189) attenuated in C. elegans killing

27. ace189 microscopy BG2 ace189

28. ace189 genetic characterization 5’ of Cg PAN1 homologue Pan1p (S. cerevisiae) –essential in S. cerevisiae –localizes to cortical actin patches (with End3p, Sla1p, and several clathrin assembly proteins) –binds and activates the Arp2/3 complex (actin polymerization) –involved in... endocytosis organization of the actin cytoskeleton cell wall morphology PAN1 4.4 kb PMR1 2.7 kb ATG

29. Cell wall morphology of ScPAN1 mutant Tang H-Y, et al. Mol. Bio. Cell. 20:12-25, 2000. CRY1 (wildtype)pan1-4 @ 24 o C pan1-4 @ 37 o C

30. BG2 BG14 ace189 Decreased fitness of ace189 Slide 1 SD media - 30 o C YPD - 30 o C BG2 BG14 ace189

31. BG2 BG14 ace189 Decreased fitness of ace189 Slide 2 YPD - 42 o C YPD + sorbitol - 42 o C BG2 BG14 ace189

32. Characterization of ace189/PAN1 Transform BG14 (ura3-) with YIplac211-rescue plasmid and characterize reintegratants: -Confirm homologous recombination. -Confirm nematode phenotype. -Test in immunocompromised mouse model. Construct and characterize Candida PAN1 deletion mutants: -C. glabrata. Construct CgPAN1 deletion mutant. -C. albicans. Construct pan1/pan1 homozygous deletion mutant if possible. -Test Cg and Ca deletion mutants in nematodes and mice. -Construct and test complemented mutants in nematodes and mice. -Characterize mutants: endocytosis (lucifer yellow), cell wall (EM). -Construct complements with ScPAN1 and examine cell biology (endocytosis and possibly EM).

33. Completion of C. glabrata screen Confirm final set of mutants Southern blot of mutants Plasmid rescue and sequence analysis of mutants Construct reintegrants (from plasmid rescue) of the single and tandem mutants, and retest them in worms Construct and characterize C. glabrata and C. albicans deletion mutants in worms and mice Secondary phenotype analysis

34. Acknowledgements Massachusetts General Hospital Steve Calderwood Brian Enloe Eleftherios Mylonakis Fred Ausubel Andrew Diener Jay Fishman Robert Koch-Institut, Berlin Bernard Hübe Support Howard Hughes Medical Institute National Institutes of Health Aventis S.A.