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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
Cryptococcus neoformans
Important pathogen
Some molecular biology
foundation
Significant number
of investigators
Genome sequence
in progress
Well-studied pathophysiology
Known virulence phenotypes
Excellent animal models
A number of yeasts are pathogenic
in the C. elegans model
C. laurentii
% Survival
100
R. mucilaginosa
75
50
S. cerevisiae
25
C. neoformans H99
C. parapsilosis
0
0
100
Time (hrs)
200
Lifespan of wild type C. elegans on lawns of
non-pathogenic organisms
% Survival
100
C. kuetzingii
75
C. laurentii
E. coli OP50
50
25
0
0
200
400
Time (hours)
600
C. neoformans strains from different
serogroups are pathogenic
100
% Survival
C. laurentii
75
C. neoformans serotype A
50
C. neoformans serotype D
C. neoformans serotype A
25
C. neoformans serotype B/C
0
0
100
200
Time (hrs)
300
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.
Daf-2 nematodes have a significantly longer
survival on lawns of C. neoformans
compared to N2s
(LT50: 9 vs. 4.5 days)
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.
FACS analysis and corresponding phase-contrast and
epifluorescent microscopy of MF1::GFP from CSF
Evaluation of Cryptococcal
Virulence Factors in C. elegans
GPA1 regulates capsule and melanin
production in C. neoformans
Gpa enhances virulence in C. elegans
% Survival
100
75
gpa
50
H 99
25
GPA
0
0
50
100
Time (hrs)
150
200
PKA1 gene encods the major cAMP-dependent protein
kinase catalytic subunit and PKR1 gene encods the
protein kinase A (PKA) regulatory subunit
PKA/
PKR
PKA1 enhances virulence in mice
PKA1 enhances virulence in the
C. elegans model
100
% Survival
pka
75
50
25
H 99
PKA
0
0
50
100
Time (hrs)
150
200
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)
The same pkr1 mutant is hypervirulent
in the C. elegans model
% Survival
100
75
H99
50
25
pkr
0
0
50
Time (hrs)
100
150
Melanogenesis from dopamine in C. neoformans
When DA was added in media, the lacc+ strain of
C. neoformans was more virulent than an otherwise
isogenic lacc- strain
% Survival
100
Lacc- DAlacc+ DA-
75
50
lacc- DA+
25
lacc+ DA+
0
0
50
Time (hrs)
100
Nematodes with enhanced susceptibility
to bacterial pathogens are also more
susceptible to C. neoformans
100
% Survival
H99 N2
75
H99 esp8
50
25
H99 esp2
0
0
20
40
Time (hrs)
60
C. neoformans/C. elegans model system
Summary
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
Acknowledgements
Calderwood laboratory
MGH
Rhonda Feinbaum
Dennis Kim
Kaveh Ashrafi
MIT
Gerry Fink
Robert Wheeler
Ausubel laboratory
Duke University
John R. Perfect
Joseph Heitman
John McCusker
Andrew Alspaugh
Gary M. Cox
Elizabeth A. Wills
Postdoctoral funding:
Boston University
Stu Levitz
Saint Louis University
Jennifer K. Lodge
Albert Einstein College
of Medicine
Arturo Casadevall
Howard Hughes Medical Institute