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Costi Sifri and Brian Enloe Ausubel Group Meeting April 1, 2003

Candida - C. elegans Model System. Costi Sifri and Brian Enloe Ausubel Group Meeting April 1, 2003. C. albicans , C. glabrata , C. krusei , C. parapsilosis , C. tropicalis , C. dubliniensis , and hundreds more commensal organisms of the mucous membranes

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Costi Sifri and Brian Enloe Ausubel Group Meeting April 1, 2003

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

  2. 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, Staphylococcusaureus, and enterococci) with an attributable mortality of 38%* increasing drug resistance (emergence of non-albicansCandida) Candida and Human Disease 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. eleganson modified NGM at 25oC

  4. C. elegans killing by Candida requires live yeast 100 C. albicans CAN14 - alive CAN14 - amphoB killed C. glabrata BG2 - alive BG2 - amphoB killed 75 Survival (percent) 50 25 0 0 20 40 60 80 Time (hours)

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

  6. C. elegans can be rescued from C. albicans by transfer to an innocuous food source 100 75 CAN14 CAN14-OP50 6h CAN14-OP50 18h CAN14-OP50 22h Survival (percent) 50 25 0 0 20 40 60 Time (hours)

  7. ...and may be the sole mechanism of killing by S. cerevisiae Sc : N2 Sc : glp1 Matricide is a significant component of C. elegans killing by C. albicans 100 75 Ca : N2 Ca : glp1 Survival (percent) 50 25 0 0 100 200 300 Time (hours)

  8. Characteristics of a C. elegans - Candida Model System • Rapid killing (LT50@ 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

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

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

  14. The Efg1 and Cph1 filamentation signaling pathways are important for virulence in mice Lo HJ, et al. Cell 90:939-49, 1997 105 105 107 107 105 107 105 107

  15. COMPLEMENTS efg1 / EFG1 efg1 cph1 / EFG1 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 100 75 Survival (percent) 50 25 0 0 50 100 Time (survival)

  16. C. albicans does not filament within the nematode intestine Anterior CAN14 (wt) CAN34 (efg1/efg1cph1/cph1)

  17. C. albicans does not filament within the nematode intestine Posterior CAN14 (wt) CAN34 (efg1/efg1cph1/cph1)

  18. C. albicans does not filament within the nematode intestine Close-up CAN14 (wt) CAN34 (efg1/efg1cph1/cph1)

  19. Secreted Aspartic Proteinase (SAP) mutants are not attenuated in Candida-mediated C. elegans killing Efg1 regulates SAP4, SAP5, and SAP6 gene expression 100 Wild type Ca Dsap1-3 Dsap4-6 75 Survival (percent) 50 25 0 0 20 40 60 Time (hours)

  20. Inhibition of SAPs with Pepstatin A does not alter Candida-mediated C. elegans killing 100 CAN14 (wildtype) CAN14 : pepA CAN34 (efg1 cph1) 75 Survival (percent) 50 25 0 0 20 40 60 80 Time (hours)

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

  22. Ras1 and Tup1 are not important in C. elegans killing 100 wild-type ras1 tup1 75 Survival (percent) 50 25 0 0 20 40 60 Time (survival)

  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/mg Cormack BP, et al. Science 285:578-82, 1999. Cormack BP, el al. Genetics 151:979-87, 1999.

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

  26. A representative C. glabrata mutant (ace189) attenuated in C. elegans killing 100 75 BG2 ace189 Survival (percent) 50 25 0 0 20 40 60 Time (hours)

  27. BG2 ace189 ace189 microscopy

  28. ATG ace189 genetic characterization PMR1 2.7 kb PAN1 4.4 kb • 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

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

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

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

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

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