Zakhikany et al. data

In vivo transcript profiling of Candida albicans identifies a gene essential for interepithelial dissemination

Katherina Zakikhany¹, Julian R. Naglik², Andrea Schmidt-Westhausen³, Gudrun Holland⁴, Martin Schaller⁵, and Bernhard Hube^1/6^*

¹Division Mycology, Robert Koch Institute, Germany;

²Department of Oral Immunology, King´s College London, UK;

³Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Germany;

⁴Center for Biological Safety, Robert Koch Institute, Germany;

⁵Department of Dermatology, University of Tübingen, Germany,

⁶Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural

Product Research and Infection Biology – Hans-Knöll-Institute Jena, Germany.

Summary

Candida albicans is the most common oral fungal pathogen of humans, but the mechanisms by which C. albicans invades and persists within mucosal epithelium are not clear. To understand oral pathogenesis, we characterized the cellular and molecular mechanisms of epithelial-fungus interactions using reconstituted human oral epithelium (RHE). We observed that hyphal formation facilitates epithelial invasion via both active (physical penetration) and passive (induced endocytosis) processes. Genome wide transcript profiling of C. albicans experimental RHE infection was compared with that from eleven patient samples with pseudomembranous candidiasis to identify genes associated with disease development in vivo. Expression profiles reflected the morphological switch and an adaptive response to neutral pH, non-glucose carbon sources and nitrosative stress. We identified several novel infection-associated genes with unknown function. One gene, up regulated in both RHE infection and patients, named EED1, was essential for maintenance of hyphal elongation. Mutants lacking EED1 showed transient cell elongation on epithelial tissue, which enabled only superficial invasion of epithelial cells. Once inside an epithelial cell, Δeed1 cells could proliferate as yeasts or pseudohyphae but remained trapped intracellularly. Our results suggest that the adaptive response and morphology of C. albicans play specific roles for host-fungal interactions during mucosal infections.

Key words: oral infections, patient samples, hyphal formation, induced endocytosis

These data were provided by Katherina Zakikhany and Bernhard Hube, Robert Koch-Institut, Berlin, Germany.

Experimental Data I (in vitro transcriptional profiling, RHE time course infection)

Species	Candida albicans
Strains	Wildtype: SC5314 (Gillum et al., 1984)
Growth Conditions	Experimental epithelial tissue infection (RHE), timecourse: 1-24 h p.i., preculture: YPD
RNA extraction	Harvested cells were frozen in liquid N₂. RNA was extracted using RNApure (Peqlab) following the manual.
Labelling	Direct labelling with Cy3/Cy5-dUTP as described, using the Agilent Fluorescent Linear Amplification Kit.
Reference RNA (refRNA)	The reference (Cy3) on each array was from a pool of SC5314 (Gillum et al., 1984) RNA samples from cells grown in YPD at 37°C to mid-exponential phase.
Microarray	Glass microarrays with probes for 5907 C. albicans ORFs as described.
Microarray Manufacturer	Eurogentec SA
Hybridisation Method	DIG EasyHyb hybridisation
Imaging & Data Capture	GenePix 4000B Scanner (Axon Instruments, now Molecular Devices). Software: GenePix Pro 4.1 (Molecular Devices).
Normalisation Method	Intensity dependent (Lowess) normalisation by GeneSpring 7.0 (Silicon Genetics).
Quality controll steps	Biological replicates
Statistical Analysis	GeneSpring

Microarray Data I (in vitro RHE time course infection)

Sample	Cy3 Labelling	Cy5 Labelling	Microarray Batch No.	Microarray Slide No.	Image Files (.tif)	GenePix Results file (.gpr)	Data from GeneSpring (.xls)
S1 (IV) (1h)	Common reference	SC5314	H210C	213	S1 (IV)_532 SI (IV)_635	S1 (IV)	I (A) RHE time-course- all genes I (B) RHE time course infection- all samples
S1 (V) (1h)	Common reference	SC5314	H210C	20	S1 (V)_532 S1 (V)_635	S1 (V)
S3 (IV)(3h)	Common reference	SC5314	H210	13 (stripped)	S3 (IV)_532 S3 (IV)_635	S3 (IV)
S3 (V)(3h)	Common reference	SC5314	H210C	18	S3 (V)_532 S3 (V)_635	S3 (V)
S6(I)(6h)	Common reference	SC5314	H130C	11	S6(I)_532 S6(I)_635	S6(I)
S6(II)(6h)	Common reference	SC5314	H130C	11(stripped)	S6(II)_532 S6(II)_635	S6(II)
S6(VI)(6h)	Common reference	SC5314	H210C	4	S6(VI)_532 S6(VI)_635	S6(VI)
S12 (II2)(12h)	Common reference	SC5314	H130C	7	S12 (II 2)_532 S12 (II 2)_635	S12 (II 2)
S12 (II3)(12h)	Common reference	SC5314	H130C	9	S12 (II3)_532 S12 (II3)_635	S12 (II3)
S12(III)(12h)	Common reference	SC5314	J160C	6	S12(III)_532 S12(III)_635	S12(III)
S24(II)(24h)	Common reference	SC5314	H1360C	4	S24(II)_532 S24(II)_635	S24(II)
S24.2 (IV)(24h)	Common reference	SC5314	K0130C	20	S24.2 (IV)_532 S24.2 (IV)_635	S24.2 (IV)
S24(X)(24 h)	Common reference	SC5314	F280C	32	S24(X)_532 S24(X)_635	S24(X)
S24 (XI)(24h)	Common reference	SC5314	F280C	32 (stripped)	S24 (XI)_532 S24 (XI)_635	S24 (XI)
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Experimental Data II (in vivo transcriptional profiling, patient samples)

Species	Candida albicans
Strains	C. albicans ssp.
Growth Conditions	in vivo: swabs from the oral cavity of patients infected with an oral Candidosis
RNA extraction	Patient samples (isolated from the patient with an cotton stick) were frozen in liquid N₂. RNA was extracted using RNApure (Peqlab) following the manual.
Labelling	Direct labelling with Cy3/Cy5-dUTP as described here using the Agilent Fluorescent Linear Amplification Kit.
Reference RNA (refRNA)	The reference (Cy3) on each array was from a pool of SC5314 (Gillum et al., 1984) RNA samples from cells grown in YPD at 37°C to mid-exponential phase.
Microarray	Glass microarrays with probes for 5907 C. albicans ORFs as described.
Microarray Manufacturer	Eurogentec SA
Hybridisation Method	DIG EasyHyb hybridisation
Imaging & Data Capture	GenePix 4000B Scanner (Axon Instruments, now Molecular Devices). Software: GenePix Pro 4.1 (Molecular Devices).
Normalisation Method	Intensity dependent (Lowess) normalisation by GeneSpring 7.0 (Silicon Genetics).
Quality controll steps	Biological patient samples
Statistical Analysis	GeneSpring

Microarray Data II (in vivo patient samples)

Sample	Cy3 Labelling	Cy5 Labelling	Microarray Batch No.	Microarray Slide No.	Image Files (.tif)	GenePix Results file (.gpr)	Data from GeneSpring (.xls)
J1	Common reference	Patient -sample RNA	H130C	8	J1	J1_532 J1_635	II (A) All patients: all genes II (B) Patients – all genes - pooled
J2	Common reference	Patient -sample RNA	G300C	13	J2	J2_532 J2_635
J3	Common reference	Patient -sample RNA	H130C	6	J3	J3_532 J3_635
J4	Common reference	Patient -sample RNA	G300C	9	J4	J4_532 J4_635
19	Common reference	Patient -sample RNA	J160C	4	19	19_532 19_635
25	Common reference	Patient -sample RNA	J160C	3	25	25_532 25_635
28	Common reference	Patient -sample RNA	J160C	4	28	28_532 28_635
30	Common reference	Patient -sample RNA	H210C	15	30	30_532 30_635
32	Common reference	Patient -sample RNA	H210C	15	32	32_532 32_635
34	Common reference	Patient -sample RNA	F280C	31	34	34_532 34_635
38	Common reference	Patient -sample RNA	F280C	33	38	38_532 38_635
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GF2 laboratories home pages

Useful links

Christophe d'Enfert
Fungal Biology and Pathogenicity
Institut Pasteur
25, rue du Docteur Roux
75015 Paris, France
gf2@pasteur.fr

	Galar Fungail 2
Interaction of fungal pathogens with host cells: a post-genomic approach

Microarray Data I (in vitro RHE time course infection)

Microarray Data II (in vivo patient samples)

GF2 laboratories home pages

Useful links

Christophe d'Enfert Fungal Biology and Pathogenicity Institut Pasteur 25, rue du Docteur Roux 75015 Paris, France gf2@pasteur.fr

Christophe d'Enfert
Fungal Biology and Pathogenicity
Institut Pasteur
25, rue du Docteur Roux
75015 Paris, France
gf2@pasteur.fr