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 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 Uganda, among HIV-infected adults cryptococcal disease is diagnosed in 4040/100,000 person-years and is associated with 17% of all deaths
In developing countries, t
he mortality during
initial hospitalization
can exceed 40%.

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 50
E. coli OP50 25
200
400
600
Time (hours)

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

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

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

15. % Survival Time (hours) 100 Dpka 75 50 Dcap59 H99 25 D-pkr 50 100 15050
100
150
200
Time (hours)

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

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

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

21. 100 75
cap59-
% Survival 50
H99 25 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. % Survival Time (hours) 50 100 150 200 1B-A10 4B-B7 H99 4B-D8 100 7525
4B-D8 50
100
150
200
Time (hours)

26. Flanking regions of insertional mutants with loss-of-virulence in C
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 on N. crassa or S. pombe settings.

29. % Survival Time (hours) MYA-419 MYA-420 MYA-422 100 MYA-419 75 5025
MYA-422 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 utilize the 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. Postdoctoral funding: Howard Hughes Medical Institute
Acknowledgements
Calderwood laboratory
Ausubel 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

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

35. % Survival Time (hours) L4440 pik ikb 177 1.00 0.75 0.50 0.25 0.00 5050
100
150
Time (hours)

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