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