Costi Sifri and

1
Candida - C. elegans Model System

• Costi Sifri and
• Brian Enloe
• Ausubel Group Meeting
• April 1, 2003

2
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. 1482349-53,
1988. Edmond MB, et al. Clin. Infect. Dis.
29239-44, 1999.
3
Clinical Candida isolates kill C. eleganson
modified NGM at 25oC
4
C. elegans killing by Candida requires live yeast
100
C. albicans CAN14 - alive CAN14 - amphoB
killed C. glabrata BG2 - alive BG2 -
amphoB killed
75
Survival (percent)
50
25
0
0
20
40
60
80
Time (hours)
5
Candida accumulates within the C. elegans
intestine
C. albicans/GFP - 20 hour incubation
6
C. elegans can be rescued from C. albicans by
transfer to an innocuous food source
100
75
CAN14 CAN14-OP50 6h CAN14-OP50 18h CAN14-OP50 22h
Survival (percent)
50
25
0
0
20
40
60
Time (hours)
7
Matricide is a significant component of C.
elegans killing by C. albicans
100
75
Ca N2 Ca glp1
Survival (percent)
50
25
0
0
100
200
300
Time (hours)
8
Characteristics of a C. elegans - Candida Model
System

• Rapid killing (LT50 _at_ 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 gt TSA gt BHI
• Temperature dependant
• All stages are killed Young Adults gt L4s gt L1s
• Young larvae do not grow or mature on Candida

9
Candida accumulates within the L1 intestine but
does not support worm maturation and growth
L1 larvae on C. glabrata - 48 hour incubation
10
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

11
Invasion of esophageal tissue with C. albicans
12
Candida albicans Filamentation Pathway
?
?
Ras1
Cyr1
MAP kinase cascade
cAMP
CELL MORPHOLOGY
Tup1
Efg1
Cph1
GENE EXPRESSION
13
Morphology of C. albicans wildtype and efg1-cph1
mutant
CAN14 wild-type
CAN34 efg1/efg1 cph1/cph1
14
The Efg1 and Cph1 filamentation signaling
pathways are important for virulence in mice
Lo HJ, et al. Cell 90939-49, 1997
105
105
107
107
105
107
105
107
15
The Efg1 and Cph1 filamentation signaling
pathways are important in C. elegans killing
Cph1 MAP kinase pathway Efg1 cAMP
PKA pathway
wild-type cph1 efg1 efg1 cph1
100
75
Survival (percent)
50
25
0
0
50
100
Time (survival)
16
C. albicans does not filament within the nematode
intestine
Anterior
CAN14 (wt)
CAN34 (efg1/efg1 cph1/cph1)
17
C. albicans does not filament within the nematode
intestine
Posterior
CAN14 (wt)
CAN34 (efg1/efg1 cph1/cph1)
18
C. albicans does not filament within the nematode
intestine
Close-up
CAN14 (wt)
CAN34 (efg1/efg1 cph1/cph1)
19
Secreted Aspartic Proteinase (SAP) mutants are
not attenuated in Candida-mediated C. elegans
killing Efg1 regulates SAP4, SAP5, and SAP6 gene
expression
100
Wild type Ca Dsap1-3 Dsap4-6
75
Survival (percent)
50
25
0
0
20
40
60
Time (hours)
20
Inhibition of SAPs with Pepstatin A does
not alter Candida-mediated C. elegans killing
100
CAN14 (wildtype) CAN14 pepA CAN34 (efg1 cph1)
75
Survival (percent)
50
25
0
0
20
40
60
80
Time (hours)
21
Candida albicans Filamentation Pathway
?
?
Ras1
Cyr1
MAP kinase cascade
cAMP
CELL MORPHOLOGY
Tup1
Efg1
Cph1
GENE EXPRESSION
22
Ras1 and Tup1 are not important in C. elegans
killing
100
wild-type ras1 tup1
75
Survival (percent)

50
25
0
0
20
40
60
Time (survival)
23
Nematodes are able to consume filamentous Candida
CAN39 (tup1/tup1)
24
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/mg
Cormack BP, et al. Science 285578-82,
1999. Cormack BP, el al. Genetics 151979-87,
1999.
25
Candida glabrata screen
Primary 1028 mutants
26
A representative C. glabrata mutant
(ace189) attenuated in C. elegans killing
100
75
BG2 ace189
Survival (percent)
50
25
0
0
20
40
60
Time (hours)
27
ace189 microscopy
28
ace189 genetic characterization
PAN1 4.4 kb
PMR1 2.7 kb

• 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

29
Cell wall morphology of ScPAN1 mutant
CRY1 (wildtype)
pan1-4 _at_ 24oC
pan1-4 _at_ 37oC
Tang H-Y, et al. Mol. Bio. Cell. 2012-25, 2000.
30
Decreased fitness of ace189 Slide 1
BG2
BG2
BG14
BG14
ace189
ace189
SD media - 30oC
YPD - 30oC
31
Decreased fitness of ace189 Slide 2
BG2
BG2
BG14
BG14
ace189
ace189
YPD - 42oC
YPD sorbitol - 42oC
32
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).

33
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

34
Acknowledgements

• Massachusetts General Hospital
• Steve Calderwood
• Brian Enloe
• Eleftherios Mylonakis
• Fred Ausubel
• Andrew Diener
• Jay Fishman

• Robert Koch-Institut, Berlin
• Bernard Hübe

Support Howard Hughes Medical Institute National
Institutes of Health Aventis S.A.