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

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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

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CAN14 CAN14-OP50 6hCAN14-OP50 18hCAN14-OP50 22h

C. elegans can be rescued from C. albicans by transfer to an innocuous food source

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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

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Lo HJ, et al. Cell 90:939-49, 1997

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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

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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

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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

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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

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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.