Candida albicans and the

ICL1 gene
constructing a double knock-out

Matthijs Dekkers
Supervisor: Els Mol

Mei/Juni 2005

Introduction

Candida albicans, a

fungal pathogen that

causes opportunistic
infections
Systemic fungal infections
in immunocompromised
Increased prevalence and
severity with rise of AIDS,
chemotherapy, organ
transplantation

Introduction

Candida is the focus

of many studies
Understanding
pathogenesis
Finding new targets
for antifungals

Dimorphism
Ability to switch
between yeast and
hyphal growth
Essential for its
pathogenicity
Allows escape from
macrophage

Phagocytosis

C. albicans is phagocytosed by macrophages

and neutrophils
Normally destroyed in the phagolysosome
C. albicans can use different carbon sources, by
rapidly adapting its metabolism
This allows C. albicans to grow hyphae and
perforate the plasma membrane of the
macrophage
Releases the fungal cell while killing the
macrophage

Glyoxylate Cycle
Glucose is the
preferred carbon
source in most
organisms
Glyoxylate cycle
allows use of C2
carbon compounds
Studies have shown
that genes are
upregulated after
phagocytosis

Isocitrate Lyase

Isocitrate lyase, product of

the CaICL1 gene, is a key
enzyme
Hydrolyzes isocitrate (C6)
to succinate (C4)
Activity both specific and
limited to glyoxylate cycle
ICL1 shares homology
with other fungi, plants,
bacteria but not mammals
This makes it a possible
target for antifungal
agents

Aim of this Study

To allow investigation of the role of
isocitrate lyase in the pathogenicity of C.
albicans, in this study deletion mutants for
the ICL1 gene will be constructed.
PCR-based gene targeting involving
homologous recombination will be used to
create a double knock-out strain of C.
albicans.

Materials & Methods

PCR-based Gene Manipulation

PCR amplification of
cassettes containing
markers (ARG4 or HIS1)
Primers have 80 bases
corresponding with ICL1 at
its 5 end 20 bases with the
3 end of the cassette
PCR results in a construct
with a marker gene and
80bp homology with ICL1
on both ends
A second PCR with 38b
overlapping primers ensures
homology

ICL1

ARG4

ARG4

ARG4

PCR-based Gene Manipulation

When introduced to Candida so that
homologous recombination occurs, the
marker gene replaces the target gene
(ICL1), resulting in a single knock-out
A second knock-out will be done in the
same way, using a different marker

PCR-based Gene Manipulation

ICL1

ARG4
PCR

ARG4
recombination

ARG4

BWP-17

BWP17 strain bears 3

deletions in
auxotrophic genes:
URA3, HIS1, ARG4
allowing selection for
transformants on
plates lacking
histidine or arginine

Verification of Transformants
PCR technique will be used to verify the
correct integration of the marker and thus
the knock-out
different combinations of marker specific
and ICL1 specific primers will give
definitive answer whether transformants
are ICL1 knock-outs

Verification of Transformants
FP

A4

ARG4
A5

RP
PCR

FP+A4

FP+RP

A5+RP

Results

Cassette amplification

Amplification of ARG4
and HIS1 Wendland
cassettes with ICL1
homologous primers
results in 1900bp and
1428bp fragments

Cassette amplification
Amplification of ARG4
and HIS1 Wendland
cassettes with ICL1
homologous primers
results in 1900bp and
1428bp fragments
PCR products will be
elongated and used in
transformation

HIS1
ARG4

1900bp

1400bp

First Transformation
2 transformations, 1
with HIS1, 1 with
ARG4 were done on
BWP17 and plated on
resp. SD -his and SD
-arg plates
resulting
transformants were
analyzed with PCR

First Transformation
FP

A4

ARG4
A5

RP
PCR

+ARG4 +ARG4 +HIS1 +HIS1

BWP17

First Transformation
FP

A4

ARG4
A5

RP
PCR

1536bp

+ARG4 +ARG4 +HIS1 +HIS1

BWP17

First Transformation
FP

A4

ARG4
A5

RP
PCR

+ARG4 +ARG4 +HIS1 +HIS1

BWP17
789bp

First Transformation
FP

A4

ARG4
A5

RP
PCR

+ARG4 +ARG4 +HIS1 +HIS1

ARG4:

2369

ICL1:

2152

BWP17

First Transformation
CMD1 and CMD2
were both verified
by PCR to be
icl1::ARG4 /ICL1
These will be used
for further
experiments in this
study

CMD1

icl1::ARG4 /ICL1

CMD2

Second Transformation
The second transformation proved to be
harder than the first
Histidine cassette integrated in genome,
but not in ICL1 locus
A new screening technique was used

A Different Approach

Transformants lacking
both ICL1 alleles should
show attenuated growth
on ethanol/acetate plates
All double transformants
were plated on:

YPD
YNB + 2% glucose
YNB + 2% acetate
YNB + 2% ethanol

(+uri, +his, +arg)

A Double Knock-Out?

1 transformant did not grow at all, both on

acetate and ethanol plates, but showed normal
growth on YPD and YNB + glucose. This strain
was further analyzed

YPD

YNB + 2% acetate

Second Transformation
FP

A4

ARG4
A5

RP
PCR

1536bp

CMD1

CMD3

BWP17

Second Transformation
FP

A4

ARG4
A5

RP
PCR

CMD1

CMD3

BWP17

789bp

Second Transformation
FP

H5

HIS1
H4

RP
PCR

CMD1

CMD3

BWP17

415bp

Second Transformation
FP

H5

HIS1
H4

RP
PCR

400bp

CMD1

CMD3

BWP17

Second Transformation
FP

HIS1
RP

FP

ARG4
PCR

2369bp

CMD1

CMD3

RP

BWP17

1781bp

A Double Knock-Out

CMD3 was verified by

selection on arg, -his
plates and by PCR to be

icl1::ARG4 /icl1::HIS1

CMD3

icl1::ARG4 /icl1::HIS1

Future Research
Complementation of CMD3 with ICL1
Growth comparison on different media

wild type
BWP17
CMD1, CMD2 and CMD3
complemented CMD3

Virulence tests in mice

Acknowledgements
thanks to everybody, especially
Els Mol

Questions & Discussion