Candida albicans and the icl1 gene
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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

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Candida albicans and the ICL1 gene

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Candida albicans and the icl1 gene

Candida albicans and the ICL1 gene

constructing a double knock-out

Matthijs Dekkers

Supervisor: Els Mol

Mei/Juni 2005


Introduction

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


Introduction1

Introduction

  • Candida is the focus of many studies

  • Understanding pathogenesis

  • Finding new targets for antifungals


Dimorphism

Dimorphism

  • Ability to switch between yeast and hyphal growth

  • Essential for its pathogenicity

  • Allows escape from macrophage


Phagocytosis

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

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

  • 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

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

Materials & Methods


Pcr based gene manipulation

ICL1

ARG4

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

ARG4

ARG4


Pcr based gene manipulation1

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 manipulation2

PCR

ARG4

PCR-based Gene Manipulation

ICL1

ARG4

ARG4

recombination


Bwp 17

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

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 transformants1

FP

A4

PCR

A5

RP

ARG4

Verification of Transformants

FP+A4

A5+RP

FP+RP


Results

Results


Cassette amplification

Cassette amplification

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


Cassette amplification1

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

±1400bp

ARG4

±1900bp


First transformation

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 transformation1

First Transformation

FP

A4

ARG4

A5

RP

PCR

+ARG4

+ARG4

+HIS1

+HIS1

BWP17


First transformation2

First Transformation

FP

A4

ARG4

A5

RP

PCR

+ARG4

+ARG4

+HIS1

+HIS1

BWP17

1536bp


First transformation3

First Transformation

FP

A4

ARG4

A5

RP

PCR

+ARG4

+ARG4

+HIS1

+HIS1

BWP17

789bp


First transformation4

First Transformation

FP

A4

ARG4

A5

RP

PCR

+ARG4

+ARG4

+HIS1

+HIS1

BWP17

ARG4: 2369

ICL1:2152


First transformation5

CMD1

CMD2

icl1Δ::ARG4 /ICL1

First Transformation

  • CMD1 and CMD2 were both verified by PCR to be icl1Δ::ARG4 /ICL1

  • These will be used for further experiments in this study


Second transformation

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

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

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

YNB + 2% acetate

YPD


Second transformation1

Second Transformation

FP

A4

ARG4

A5

RP

PCR

CMD1

CMD3

BWP17

1536bp


Second transformation2

Second Transformation

FP

A4

ARG4

A5

RP

PCR

CMD1

CMD3

BWP17

789bp


Second transformation3

H5

H4

RP

Second Transformation

FP

HIS1

HIS1

PCR

CMD1

CMD3

BWP17

415bp


Second transformation4

H5

H4

RP

Second Transformation

FP

HIS1

HIS1

PCR

400bp

CMD1

CMD3

BWP17


Second transformation5

RP

Second Transformation

FP

HIS1

FP

ARG4

PCR

RP

2369bp

CMD1

1781bp

CMD3

BWP17


A double knock out1

A Double Knock-Out

  • CMD3 was verified by selection on –arg, -his plates and by PCR to be icl1Δ::ARG4 /icl1Δ::HIS1

CMD3

icl1Δ::ARG4 /icl1Δ::HIS1


Future research

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

Acknowledgements

thanks to everybody, especially Els Mol


Questions discussion

Questions & Discussion


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