Study of Cryptococcus neoformans Pathogenesis using Multihost System

1. Using the “multihost system” for the study of the pathogenesis of the model yeast C. neoformans

2. Cryptococcus neoformans Most often affects the CNS, lungs or the skin • Predisposing factors: HIV, steroid therapy, transplantation and malnutrtion Rates in patients without HIV infection in the U.S. comparable with the incidence rates of meningococcal meningitis. The annual incidence of cryptococcosis among HIV-infected patients was 1638.7 per 100,000 individuals

3. Cryptococcus neoformans There has been an explosion in the incidence of cryptococcosis in Africa, Thailand, and India Cryptococcal meningitis is the leading cause of culture-positive meningitis in Zimbabwe, constituting 45% of all cases. In developing countries, t he mortality during initial hospitalization can exceed 40%. In Uganda, among HIV-infected adults cryptococcal disease is diagnosed in 4040/100,000 person-years and is associated with 17% of all deaths

4. Cryptococcus neoformans • Among immunocompromised patients, cryptococcal infection is caused by C. neoformans var. neoformans while the other variety, C. neoformans var. gattii is associated with infections among immunocompetent individuals in tropical and subtropical areas and especially in Australia and New Guinea, where it grows on Eucalyptus trees.

5. Cryptococcus neoformans • A significant epidemic caused by C. neoformans var. gattii on Vancouver Island has challenged this understanding. A total of 59 cases were reported between January 1999 and August 2002. • At least 64% of the cases involved immunocompetent individuals and 24% developed meningitis with at least 6 deaths. • Eucalyptus trees did not show evidence of the organism.

6. Lifespan of wild type C. elegans on lawns of non-pathogenic organisms 100 C. kuetzingii 75 C. laurentii % Survival E. coli OP50 50 25 0 0 200 400 600 Time (hours)

12. 100 Agar 75 % Survival 50 25 0 0 50 100 150 Time (hours)

13. 1.00 cap59-HK kuetz-HK 0.75 H99-HK % Survival 0.50 0.25 0.00 0 50 100 150 Time (hours)

14. Kaplan-Meier survival estimates, by Group 1.00 0.75 0.50 0.25 GPA-R 0.00 0 50 100 150 200 analysis time dgpa dpka H99 PKA-R dpkr

15. 100 Dpka 75 % Survival Dcap59 50 H99 D-pkr 25 0 0 50 100 150 200 Time (hours)

16. 100 75 H99-Food % Survival 50 H99 25 0 0 50 100 150 200 Time (hours)

19. 1.00 0.75 % Survival 0.50 OR imd/spz 0.25 0.00 0 50 100 150 Time (hours)

20. 100 OR 75 % Survival 50 25 spz- 0 0 50 100 150 Time (hours)

21. 100 75 cap59- % Survival 50 H99 25 0 0 50 100 Time (hours)

22. Random insertion mutagenesis I • C. neoformans strain H99 • The yeast cells were transformed with DNA-coated gold beads using a particle gun. • Plasmid pCH233, which features the neosourthricin resistance gene flanked by the actin promoter • Stable transformants were checked for melanin production, growth at 37C, growth on minimal medium and capsule production

23. Random insertion mutagenesis II • Stain F99 is a spontaneous 5-FOA resistant (Ura-) derivative of strain H99, which is virulent in both worm and vertebrate models of infection. The strain contains a point mutation in the URA5 gene. • Plasmid pJMM97-3DRI carries the wild type URA5 gene, otherwise the vector has no regions of homology to the C. neoformans genome. • Biolostic transformation of F99 with KpnI linerized pJMM97-3DRI resulted in a transformation efficiency of approximately 50 to 100 transformants per microgram.

24. F99 Ura+ transformants URA5 3 kb 2 kb 1.5 kb 1 kb 0.5 kb Of 16 transformants tested, 16 were stable, suggesting genomic integration. Ten of these were shown to contain random single integrations, one contained two independent random integrations, two showed gene conversions at the URA5 locus, and three were of ambiguous genetic structure. Strains were tested by Southern blot. Genomic DNA digested with XhoI, which cuts once in pJMM97-3DRI, and probed with full length vector.

25. 100 1B-A10 75 % Survival 4B-B7 50 H99 4B-D8 25 0 0 50 100 150 200 Time (hours)

26. Flanking regions of insertional mutants with loss-of-virulence in C. elegans • Contigs refer to H99 contigs from Duke sequencing. • Gene prediction using FGENESH at www.softberry.com on N. crassa or S. pombe settings.

29. 100 MYA-419 MYA-419 75 % Survival MYA-420 50 MYA-420 MYA-422 25 MYA-422 0 0 50 100 150 200 Time (hours)

30. Strategy for targeted disruption of genes involved in iron metabolism

32. C. neoformans/C. elegans model system Summary C. elegans can utilizethe nonpathogenic 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 This model system can be used to rapidly screen for new genes important in virulence, which may lead to new targets for antifungal therapies.

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

34. N2 100 pik ikb 75 esp2 esp8 % Survival 50 N2 25 pik esp8 esp2 ikb 0 0 100 200 300 Time (hours)

35. 1.00 0.75 L4440 pik % Survival 0.50 ikb 177 0.25 0.00 0 50 100 150 Time (hours)

36. 1.00 0.75 N2 % Survival 0.50 PIK NON-GREEN EM3 PIK 0.25 PIK GREEN EM3 0.00 0 50 100 150 200 Time (hours)