2. Cryptococcus neoformans Basidiomycete yeast Both an opportunistic and a primary pathogen mainly affecting CNS Serotype D More frequent in Europe Serotype A Worldwide distribution Serotypes B and C (var. gattii): Tropical and subtropical regions Primarily infects hosts with normal immune function

3. Cryptococcus neoformans Important pathogenSome molecular biology foundation Well-studied pathophysiology Known virulence phenotypes Excellent animal models Significant number of investigators Genome sequence in progress

4. 0100200 0 25 50 75 100 C. parapsilosis C. laurentii C. neoformans H99 R. mucilaginosa S. cerevisiae % Survival Time (hrs) A number of yeasts are pathogenic in the C. elegans model

5. % Survival Time (hours) 0200400600 0 25 50 75 100 C. kuetzingii E. coli OP50 C. laurentii Lifespan of wild type C. elegans on lawns of non-pathogenic organisms

6. C. neoformans strains from different serogroups are pathogenic Time (hrs) % Survival 0100200300 0 25 50 75 100 C. laurentii C. neoformans serotype B/C C. neoformans serotype D C. neoformans serotype A

8. Killing is not solely a matricidal effect: Glp-4 nematodes die approximately 2 days later than N2 when placed on lawns of C. neoformans, but their lifespan is significantly shorter than on lawns of nonpathogenic cryptococci C. neoformans does not colonize C. elegans: N2 nematodes were fed on lawns of C. neoformans and then at different time-points transferred to lawns of non-pathogenic cryptococci. All nematodes that survived transfer were rescued, regained normal non-distended anatomy and no C. neoformans yeast cells could be cultured followed “grinding” of the rescued worms.

9. Daf-2 nematodes have a significantly longer survival on lawns of C. neoformans compared to N2s (LT 50 : 9 vs. 4.5 days)

10. Expression of Cryptococcal Virulence Genes inside C. elegans The MAT mating-type alpha locus containing the MFalpha gene has been linked with virulence Cryptococcal cells containing the promoter of MFalpha1 fused to GFP documented the expression of this gene in the CNS of infected rabbits.

11. FACS analysis and corresponding phase-contrast and epifluorescent microscopy of MF1::GFP from CSF

13. Evaluation of Cryptococcal Virulence Factors in C. elegans

14. GPA1 regulates capsule and melanin production in C. neoformans

15. 050100150200 0 25 50 75 100 gpa GPA H 99 % Survival Time (hrs) Gpa enhances virulence in C. elegans

16. PKA1 gene encods the major cAMP-dependent protein kinase catalytic subunit and PKR1 gene encods the protein kinase A (PKA) regulatory subunit PKA/ PKR

17. PKA1 enhances virulence in mice

18. 050100150200 0 25 50 75 100 pka H 99 PKA % Survival Time (hrs) PKA1 enhances virulence in the C. elegans model

19. A strain with a mutation involving PKR1 is hypervirulent in animal models (A)Survival (B) Corticosteroid immunosuppressed rabbits. Quantitative cultures (C) Quantitative cultures (brain/lung/spleen)

20. 050100150 0 25 50 75 100 H99 pkr The same pkr1 mutant is hypervirulent in the C. elegans model % Survival Time (hrs)

21. Melanogenesis from dopamine in C. neoformans

22. % Survival Time (hrs) When DA was added in media, the lacc+ strain of C. neoformans was more virulent than an otherwise isogenic lacc- strain 050100 0 25 50 75 100 lacc+ DA+ lacc+ DA - lacc- DA+ Lacc- DA-

23. 0204060 0 25 50 75 100 H99 N2 H99 esp2 H99 esp8 p Nematodes with enhanced susceptibility to bacterial pathogens are also more susceptible to C. neoformans % Survival Time (hrs)

24. C. elegans can utilize the non-pathogenic yeasts C. laurentii and C. kuetzingii as a sole source of food The human pathogenic yeast C. neoformans kills C. elegans Several C. neoformans genes, previously shown to be involved in mammalian virulence, also play a role in C. elegans killing The nematode response to C. neoformans may have some interesting similarities to the response of mammals C. neoformans/C. elegans model system Summary

26. Calderwood laboratory Ausubel laboratory Acknowledgements MGH Rhonda Feinbaum Dennis Kim Kaveh Ashrafi MIT Gerry Fink Robert Wheeler Duke University John R. Perfect Joseph Heitman John McCusker Andrew Alspaugh Gary M. Cox Elizabeth A. Wills Postdoctoral funding: Howard Hughes Medical Institute Boston University Stu Levitz Saint Louis University Jennifer K. Lodge Albert Einstein College of Medicine Arturo Casadevall