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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 - PowerPoint PPT Presentation
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Costi Sifri and Brian Enloe
Ausubel Group MeetingApril 1, 2003
Candida - C. elegans Model System
Candida and Human Disease
• 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, Staphylococcus aureus, and enterococci) with an attributable mortality of 38%*
• increasing drug resistance (emergence of non-albicans Candida)
Wey SB, et al. Arch. Intern. Med. 148:2349-53, 1988.Edmond MB, et al. Clin. Infect. Dis. 29:239-44, 1999.
Isolate Species 48h mortality (%)
S510 C. albicans 98.4 2.2
W972 C. albicans 100
X669 C. glabrata 89.7 4.6
T308 C. glabrata 98.3 2.4
M293 C. tropicalis 98.9 1.6
W126 C. tropicalis 100
W322 C. krusei 100
F270 C. krusei 92.6 3.2
F429 C. parapsilosis 100
F431 C. guilliermondii 98.8 1.7
L6220 S. cerevisiae 38 14
OP50 E. coli 0
Clinical Candida isolates kill C. eleganson modified NGM at 25oC
Su
rviv
al (
perc
ent)
Time (hours)
0 20 40 60 80
0
25
50
75
100 C. albicans
CAN14 - aliveCAN14 - amphoB killed
C. glabrata
BG2 - aliveBG2 - amphoB killed
C. elegans killing by Candida requires live yeast
Candida accumulates within the C. elegans intestine
C. albicans/GFP - 20 hour incubation
Anterior Posterior
Su
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perc
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Time (hours)0 20 40 60
0
25
50
75
100
CAN14 CAN14-OP50 6hCAN14-OP50 18hCAN14-OP50 22h
C. elegans can be rescued from C. albicans by transfer to an innocuous food source
Su
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perc
ent)
Time (hours)0 100 200 300
0
25
50
75
100
Ca : N2Ca : glp1
Matricide is a significant component ofC. elegans killing by C. albicans
...and may be the sole mechanism of killing by S. cerevisiae
Sc : N2Sc : glp1
• 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
Characteristics of a C. elegans - Candida Model System
Candida accumulates within the L1 intestine but does not support worm
maturation and growth
L1 larvae on C. glabrata - 48 hour incubation
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
Invasion of esophageal tissue with C. albicans
Candida albicans Filamentation Pathway
Starvation
Ras1
MAPkinase
cascade
Cph1
CELLMORPHOLOGY
Cyr1
cAMP
Efg1
? ?
GENE EXPRESSION
Tup1
CAN14wild-type
CAN34efg1/efg1 cph1/cph1
Morphology of C. albicans wildtype and efg1-cph1 mutant
The Efg1 and Cph1 filamentation signaling pathways are important for virulence in mice
105
107
105
105
105
107
107
107
Lo HJ, et al. Cell 90:939-49, 1997
Su
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perc
ent)
Time (survival)0 50 100
0
25
50
75
100
The Efg1 and Cph1 filamentation signaling pathways are important in C. elegans killing
Cph1 = MAP kinase pathway Efg1 = cAMP PKA pathway
wild-typecph1efg1efg1 cph1
efg1 / EFG1efg1 cph1 / EFG1
COMPLEMENTS
CAN14 (wt)
CAN34 (efg1/efg1 cph1/cph1)
Anterior
C. albicans does not filament within the nematode intestine
CAN14 (wt)
CAN34 (efg1/efg1 cph1/cph1)
Posterior
C. albicans does not filament within the nematode intestine
C. albicans does not filament within the nematode intestine
CAN14 (wt)
CAN34 (efg1/efg1 cph1/cph1)
Close-up
Su
rviv
al (
perc
ent)
Time (hours)0 20 40 60
0
25
50
75
100
Wild type Casap1-3sap4-6
Secreted Aspartic Proteinase (SAP) mutantsare not attenuated in Candida-mediated
C. elegans killingEfg1 regulates SAP4, SAP5, and SAP6 gene expression
Time (hours)
0 20 40 60 80
0
25
50
75
100
Su
rviv
al (
perc
ent) CAN14 (wildtype)
CAN14 : pepACAN34 (efg1 cph1)
Inhibition of SAPs with Pepstatin A does notalter Candida-mediated C. elegans killing
Candida albicans Filamentation Pathway
Starvation
Ras1
MAPkinase
cascade
Cph1
CELLMORPHOLOGY
Cyr1
cAMP
Efg1
? ?
GENE EXPRESSION
Tup1
0 20 40 60
0
25
50
75
100
Time (survival)
Su
rviv
al (
perc
ent)
wild-typeras1tup1
Ras1 and Tup1 are notimportant in C. elegans killing
Nematodes are able to consume filamentous Candida
CAN39 (tup1/tup1)
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/g
Cormack BP, et al. Science 285:578-82, 1999.Cormack BP, el al. Genetics 151:979-87, 1999.
Primary1028 mutants
Candida glabrata screen
Secondary74 mutants
Tertiary18 of 71 are attenuated
3 left to test
Auxotrophic on NGM14 mutants
12 are attenuated
Time (hours)
0 20 40 60
0
25
50
75
100
Su
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perc
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BG2ace189
A representative C. glabrata mutant (ace189)attenuated in C. elegans killing
ace189 microscopy
BG2 ace189
ace189 genetic characterization
• 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
PAN14.4 kb
PMR12.7 kb
ATG
Cell wall morphology of ScPAN1 mutant
Tang H-Y, et al. Mol. Bio. Cell. 20:12-25, 2000.
CRY1 (wildtype) pan1-4 @ 24oC pan1-4 @ 37oC
BG2
BG14
ace189
Decreased fitness of ace189Slide 1
SD media - 30oC YPD - 30oC
BG2
BG14
ace189
BG2
BG14
ace189
Decreased fitness of ace189Slide 2
YPD - 42oC YPD + sorbitol - 42oC
BG2
BG14
ace189
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).
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
Acknowledgements
Massachusetts General HospitalMassachusetts General Hospital
Steve CalderwoodBrian EnloeEleftherios Mylonakis
Fred AusubelAndrew Diener
Jay Fishman
Robert Koch-Institut, BerlinRobert Koch-Institut, Berlin
Bernard Hübe
Support
Howard Hughes Medical InstituteHoward Hughes Medical InstituteNational Institutes of HealthNational Institutes of Health
Aventis S.A.Aventis S.A.