Qualitative changes lead to a deeper investigation of the
cell wall proteome of Candida albicans under iron limited
conditions
Jens M. Wartenberg
Bachelor Thesis
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV,
Amsterdam, The Netherlands
Summary
Candida albicans is an opportunistic pathogenic fungus which is carried by 80%
of the human population without causing infection. The fungus is able to become
virulent at certain environmental cues and cause infection which may result in
death. Immunocompromised patients are at higer risk of developing candidias.
Several antifungals have been artificially developed, but overuse led to resistance
and thus new drug-targets need to be identified. Iron is an important nutrient for
proliferation and growth in almost every microorganism, but due to its toxic
nature it is not freely accessible in the host. The human body packages iron in
proteins for transport and storage and so Candida needed to develop several
uptake systems. Proteins on the cell wall are known to be important in iron
acquisition. By making use of mass spectrometry techniques we have looked at
changes in the cell wall proteome of Candida albicans under iron limiting
conditions at pH 7.4 and pH4. Transcriptional studies indicate the upregulation of
several proteins, like the iron acquisition protein Rbt5, but we could not verify that
many changes on qualitative protein level.
Introduction
Fungal
research
is
becoming
increasingly important. Although the
current health system provides great
care of patients, immunocompromised
humans remain at high risk of
developing fungal infections and this is
still the leading cause of mortalities
among HIV-1 affected people. Candida
albicans is an opportunistic fungus
which lives and thrives in 80% of the
population without causing harmful
effects. A lot of research has been done
on this organism and several antifungals
have been developed, but overuse of
these has led to resistant strains and so
the quest for new drug-targets
continues. Cell-wall proteins of C.
albicans are one of the new targets to
develop fungistatic and fungicidal
vaccines without causing severe sideeffects.
A number of virulence factors have been
established in C. albicans which are
responsible
for
its
opportunistic
behaviour. It is capable of switching
from
a
unicelullar
budding-yeast
phenotype to an invasive multicellular
filamentous form called hyphae and
there is also an intermediate form
named pseudo-hyphae. This switching
is reversible and is induced by
environmental cues. The cell wall
contains a large repertoire of adhesion
proteins which are involved in the
formation of stable attachments to the
host-tissue. The fungus also secretes
proteases which presumably are
involved in nutrient supply, degradation
of immunoglobins and degradation of
host-barriers during invasion.
1
Candida is able to grow in different
habitats throughout the human body.
Three different forms of infection can be
distinguished: cutaneous, mucosal and
systemic. Mucosal infections can be
subdivided in oropharyngeal candidiasis
(OPC), esophageal candidiasis (EPC)
and vulvovaginal candidiasis (VVC).
OPC and EPC are the main mucosal
infections in HIV patients. This research
project will mainly focus on mucosal
infections.
For Candida to grow at different sites of
the human body it needs to adapt to the
environment it is surrounded by. Not
only is it able to withstand different pH
levels, but oxygen availability can range
from a pO2 of around 100 mm Hg to
almost anaerobic conditions. Iron is an
essential nutrient for growth and
proliferation for almost all micro
organisms. It is used in many enzymes
as a cofactor due to its favourable
reducing potential. Ferric iron is the
predominant form of iron, because
under standard conditions ferrous iron
will auto-oxidize to ferric iron. At pH 7
ferric iron forms a complex with water in
which protons are lost. This results in an
amorphous gel and so virtually no free
iron is available. The human body
solves this by packing and transporting
iron in proteins like lactoferrin, ferritin
and transferritin. The difficulty in
obtaining iron leads to a limited number
of uptake mechanisms in microorganisms. Iron acquisition can be
divided in two major groups: reductase
dependent and reductase indepent. The
solubility of ferrous iron at ~pH 7 is
much higher than that of ferric iron. A
logical way of iron uptake would be to
reduce FeIII to FeII. C. albicans has two
reducing uptake systems: high affinity
and low affinity. The latter directly takes
up ferrous iron by the FTR1 protein
which is a transmembrane permease.
The high affinity system first reduces
iron to reoxidize it again by ferroxidase.
The reason why and how this system
works is not known yet. Candida is able
to directly take up siderophores which
are small molecules capable of binding
ferric iron. Surprisingly it is not able to
produce siderophores itself but uses
those of other micro-organisms. Another
way of acquiring iron without reducing is
the direct uptake of haeme with Rbt5 a
GPI-anchored protein capable of binding
and transporting haeme out of
haemoglobin across the cell membrane.
Iron uptake systems are similar among
most micro-organisms. So there is not
only great difficulty in obtaining iron, but
as well great competition for it.
In this study we are investigating the
effects of iron limitation on the cell wall
proteome of Candida albicans. The cell
walls and cell wall proteins (CWP) of
fungi have several functions like water
retention, but also functions important
for virulence like adhesion, cell
aggregation, biofilm formation and
protection against oxygen radicals. Cell
wall proteins are also involved in the
uptake of iron. Here we show the effects
of iron limitation on the cell wall
proteome. We are looking at the first
stages of a mucosal infection in the
human body which include epithelial
adhesion and superficial penetration of
the mucosal surface in which hyphae
are involved.
Mass spectrometry
techniques are used to identify the
qualitative changes in the CWPs as a
consequence of the iron restriction
compared to untreated control cells.
Also the effects of low iron levels on the
ergosterol synthesis are analysed.
Materials and methods
Strains and growth media
The C. albicans strain used for the
experiments is SC5314, a clinical
isolate. Overnight cultures (ONC) were
made by inoculating Candida (stored at
4 C) in 20 mL YPD medium(1% Bactoyeast extract, 2% Bacto peptone, 2%
Glucose) for ~24 hours at 30 C and
constant shaking (200 rpm). Cells for
cell wall isolation were grown on plates
2
for 18 hours at 37 C. (3% agar, 5 mM
Mucin, 1.7% Yeast Nitrogen Base
without aminoacids and ammonium
sulfate, 75 mM Mopso (pH 7.4) or 55
mM Tartaric acid (pH 4), 0.09%
Glucose). Iron restricted plates were
prepared by adding 100 M of the iron
chelator
bathophenanthroline
di-
sulphonic acid(BPS) at pH 7.4 and
150 M BPS at pH 4.
Cell wall isolation
Invasive cells were harvested by
solubilizing the plates with 6 M
guanidine Thiocyanate, non-invasive
cells by washing off with Mili Q water.
Cells were harvested out of the plates
with 6 M Guanidine Thiocyanate or
washed of with Milli-Q water. A standard
cell wall isolation protocol (De Groot et
al. 2004) was used in which several
adaptations were made. Due to
problems with hyphae floating the
washing with Tris buffer was reduced to
one time and replaced by multiple
washing steps with MQ-water. Breaking
of cell walls by bead-beating was
increased and boiling with SDSextraction
buffer
containing
mercaptoethanol (8 L per 100 mg of
cell wall) was increased to four times to
get rid off all cytoplasmic proteins.
Isolated cell walls were freeze-dryed
and stored at – 20 C.
Sample preparation for MS
A standard protocol was used described
by Sosinska et al [2008]. No changes
were made.
MS identification of cell wall proteins
After the tryptic digest the cell walls
were centrifuged for 1 minute at 13000
rpm. The supernatant containing the
tryptic peptides was transferred to a new
tube and a standard protein zip-tip
protocol was performed using a C18
100 L zip-tip. Zip-tipped samples were
stored at – 18 C or 2 L were
dissolved in 23 L of 0.1% TFA for mass
spectrometry analysis. For protein
identification a tandem MS system was
used including a nano-LC column. The
eluted peptides are electrosprayed in a
Micromass quadrupole time-of-flight
mass spectrometer. Ions were selected
as described by Sosinska et al [2008].
Ergosterol level determination
Cells were washed off plates with MQwater and 2 mL were suspended in 6
mL Methanol. 6 mL of cold PetroleumBenzeen (PE) were added to the
sample and vortexed for 1 minute at
high speed. The samples were
centrifuged for 2 minutes at 3000 rpm.
The upper phase containing ergosterol
was transferred to a new tube and dryed
under a constant flow of gaseous
nitrogen. The dryed sample was
dissolved in 60 L ethanol to load on the
HPLC. As a standard for quantitation 1,
10 and 100 M ergosterol were used.
The different samples were normalized
by dry weight.
Dry weight/growth determination
Cells were harvested from soft agarose
plates and washed with Milli-Q water.
Samples are loaded in pre-weighted
eppendorfs and centrifuged for 1 minute
at 13000 rpm. The washing was
repeated and the samples were dried at
60 C.
Results
Iron Limitation leads to a reduction in
biomass
C. albicans is capable of growing under
Iron limited conditions to a certain
extent. Here we wanted to identify a
BPS concentration which leads to a
30% biomass reduction, because this
indicates a clear effect of the treatment,
but the cells remain viable. In Figure 1A
the effect of various BPS concentrations
on the biomass is shown. 100 M BPS,
which causes a 30% reduction in
3
A
100 uM BPS leads to a 30% growth reduction in Candida
albicans at pH 7.4
150 uM of BPS leads to a 30% growth reduction in
Candida albicans at pH 4
B
120
120
100
100
Dryweight (%)
Growth (%)
80
60
40
80
60
40
20
20
0
0
50μm BPS
100μm BPS
150μm BPS
125μm BPS
pH 7.4
150μm BPS
pH 4
Fig 1A. Growth diagram of Candida albicans with different concentrations of BPS at pH 7.4. Growth is represented in percentages with
pH 7.4 as the reference. 100 μM of BPS leads to a ~30% growth reduction which is the concentration used for further experiments. 1B Growth
diagram of Candida albicans with different concentrations of BPS at pH 4. Growth is represented in percentages with pH 4 as the
reference. 150 μM of BPS leads to a ~30% growth reduction which is the con-centration used for further experiments.
biomass, is used for further experiments
at pH 7.4. Figure 1B shows the same
experiment at pH 4, where 150 M BPS
leads to a 30% reduction in biomass
and is used for further experiments at
low pH.
Supplementation of iron can abolish the
BPS induced biomass reduction
In order to establish the fact that growth
reduction is attributed by the chelating of
iron and not by a toxic side effect of
BPS or the chelating of other essential
ions, iron was supplemented in the
media to restore growth. Figure 2A. and
2B. show growth restoration of C.
albicans cells when supplemented with
A
different
concentrations of Ferrous Iron
at pH 7.4 and pH 4.
Iron limitation leads to reduced
ergosterol levels in C. albicans
Iron is essential in the ergosterol
synthesis pathway, since many of its
enzymes contain iron, which is needed
for their function. Figure 3. shows the
Iron supplementation restores BPS caused growth inhibiton at pH 7.4
160
effect of iron limitation on ergosterol
levels. 100 M BPS shows a 40%
reduction of ergosterol in comparison
with control cells at pH 7.4. As a positive
control, cells were treated with
fluconazole, a widely used fungistatic
which inhibits 14α-demethylase, an
enzyme of the ergosterol synthesis
pathway. 0.5 g/mL fluconazole led to a
90% reduction of ergosterol levels.
Effects of iron limitation on the cell wall
proteome
Peptide mixtures of each sample are
loaded in an LC-MS-MS Q-tof massspectrometer and the derived peak list is
analysed by MASCOT. Table 1. and 2.
show the number of peptides found for
each identified protein at both pH 7.4
and pH 4 - a list of all proteins and
functions is found in supplementary data
table S1. The number of peptides is not
quantitative and only gives an indication
whether the protein is present or not.
Table 1. shows that Ssr1 and Sap9 are
found in normal treated cells at pH 7.4,
160
140
140
120
120
100
% growth
% growth
Iron supple me ntation re store s BPS cause d growth
inhibiton at pH 4
B
80
60
100
80
60
40
40
20
20
0
0
pH 7,4
100 μM BPS
100 μM BPS +
100 μM FeII
100 μM BPS +
300 μM FeII
100 μM BPS +
500 μM FeII
100 μM BPS +
1000 μM FeII
pH 4
150 μM BPS
150 μM BPS + 100
μM FeII
150 μM BPS + 300
μM FeII
Fig 2A. Iron supplementation graph of Candida albicans at pH 7.4. Growth is represented in percentages with pH 7.4 as the
reference culture. Different concentrations of ferrous iron are supplemented in plates containing 100 μM of BPS. Higher concentrations of
Iron restore growth and promote it. Fig 2B. Iron supplementation graph of Candida albicans at pH 4. Growth is represented in
percentages with pH 4 as the reference culture. Different concentrations of ferrous iron are supplemented in plates containing 100 μM of
BPS. Higher concentrations of Iron restore growth and promotes it.
4
F
C
d
li
(
Iron limited conditions lead to a 40% decrease in ergosterol
concentrations in Candida albicans
120
100
[Ergosterol (%)]
but not in the iron limited and the other
wy around for Als 5. At pH 4 Rbt5 as
well as Sod5 are found in the iron
depleted cells, but not in the normal
reference. The significance of these
findings is debatable and will be
discussed further.
80
60
40
20
Proteins
Als1
Als2
Als3
Als4
Als5
Cht2
Crh11
Ecm33
Mp65
Pga4
Phr1
Phr2
Pir1
Rbt5
Rhd3
Sap9
Sod4
Sod5
Ssr1
Utr2
Ywp1
2
pH7.4
1
3
pH7.4 BPS
2
2
5
1
3
3
7
1
4
1
4
4
3
3
1
5
2
2
2
1
2
2
4
6
6
3
2
2
2
1
2
2
3
7
1
2
7
2
2
1
9
2
2
7
2
4
2
3
1
1
1
9
1
2
3
3
6
1
1
2
3
1
1
3
5
3
6
3
5
2
1
2
1
Table 1. Number of peptides found for each protein
identified by MASCOT in normally treated cells and iron
depleted cells at pH 7.4 Normal pH 7.4 cells were done
three times and Iron limited cells were done twice
Proteins
Als1
Als2
Als3
Als4
Als5
Cht2
Crh11
Ecm33
Mp65
Pga4
Phr1
Phr2
Pir1
Rbt5
Rhd3
Sap9
Sod4
Sod5
Ssr1
Utr2
Ywp1
pH 4
pH 4+BPS
2
2
1
1
1
5
6
5
3
4
5
8
4
2
1
4
4
4
3
5
2
1
?
?
1
6
8
6
1
2
6
3
2
3
1
1
?
?
1
2
3
2
1
1
4
1
2
2
1
2
2
2
1
1
1
2
3
1
Table 2. Number of peptides found for each protein
identified by MASCOT in normally treated cells and iron
depleted cells at pH 4. The question marks are peptides
which MASCOT wasn’t able to distinguish based on the
peptide sequence, but at pH 4 only PHr2 is upregulated.
0
pH 7.4
Fluconazole
BPS
Figure 3. Ergosterol levels in two differently treated
Candida albicans cultures. Ergosterol levels were
determined with HPLC in iron limited conditions at pH 7.4 Iron
limitation leads to a 40% reduction of ergosterol. Fluconazole
(0.5 μg/mL) is shown as a positive controle.
Discussion/conclusion
In this study we show the effects of iron
depletion on growth, ergosterol levels
and the cell wall proteome of Candida
albicans. Iron is an essential nutrient
and growth is markedly affected when
iron is restricted using the iron chelator
BPS (Fig. 2A/B). At pH 4 a higher
concentration of BPS is needed to
reduce growth which is probably due to
the fact that there is more free iron
available than at neutral pH (Kosman,
2003). Reduction of growth by BPS is
caused by its iron chelating capabilities
only, because when supplemented with
iron, normal growth is restored and even
promoted (Fig. 3A/B). As stated iron is
an inducer of growth and proliferation,
thus the overshoot of growth at higher
concentrations is due to this fact.
Iron is used in many enzymatic
reactions because of its reducing
nature.
Ergosterol
is
a
critical
component of the fungal cell membrane
and the synthesis is a multistep process
in which iron is important. Depletion of
iron should therefore have an impact on
ergosterol levels. Figure 4. shows that
iron limitation leads to a 40% reduction
of the ergosterol concentration in the
cell membrane of Candida. Ergosterol
levels of cells treated with fluconazole –
a widely used fungistatic - are shown as
a positive control.
5
The cell wall proteome is dynamic and
adjusted to the environment the fungus
is surrounded by. The protocol used
leads to about 25 identified proteins by
MASCOT in a clean sample, but
normally fewer are identified at one
condition. This can be attributed to
overlapping peptide peaks and loss of
proteins during the washing and boiling
steps. Table 1. and 2. show the number
of peptides found for each identified
protein. At pH 7.4 several differences
can be seen. Ssr1 and Sap9 are found
only in the control cells. Ssr1 is a βglucan associated protein important for
cell wall structure and Sap9 is a
secreted aspartyl proteinase involved in
adhesion. Although not seen in the iron
deprived samples the number of
peptides found for the normal samples
are not that many and Ssr1 should be
present, but maybe at much lower
amounts in the iron limited samples as
well. The significance of these findings
is therefore not clear. Als5 is identified
once in the two iron limited samples, but
again the number of peptides is only
two. To be able to say something about
this difference the experiment might be
repeated for the iron deprived cells.
For pH 4 there are two differences
recorded: Sod5 which has a protective
role in oxidative stress and Rbt5 which
is involved in iron acquisition. Iron is
cytotoxic and especially auto-oxidation
of ferrous iron results in the formation of
superoxide radicals. At pH 4 more free
ferrous iron is available and so Sod5
should probably be seen in both normal
and iron limited samples. The fact that
Rbt5 is seen only in the affected
samples gives an indication that Rbt5 is
up-regulated
upon
iron
limited
conditions. This indication should be
verified by repeating the experiment and
in the future take a quantitative look at
the cell wall proteome.
Although there are some qualitative
changes on the cell wall proteome of
Candida albicans under iron restricted
conditions, the significance of these
findings have to be supported by further
experiments.
Future prospects
In this research a 30% reduction of
growth is chosen by treatment with BPS.
The effect of iron limitation on the cell
wall proteome could be more at higher
concentrations. It might be interesting to
repeat the experiments at a BPS
concentration which leads to a 50%
reduction of growth.
Although qualitatively there is no real
significant change in the cell wall
proteome of Candida albicans under
iron limitation, quantitatively it might.
The next logical step is to design an
experiment by which one can quantify
the proteins of the cell wall by making
use of mass spectrometry based
techniques. Significant down or upregulated cell wall proteins could then
be used as new potential drug-targets.
Not only iron is essential for growth and
proliferation, but oxygen availability is
also a key component in many reactions
for example the ergosterol synthesis
pathway. Sosinska et al [2008] have
looked at the effect of hypoxic
conditions
under
vagina-simulative
conditions and shown – using
immunoblot assays - an up-regulation of
Pir1 and Hwp1, but also Pga10 and
Rbt5 which are involved in iron
acquisition. Addition of the iron chelator
ferrozine led to even higher upregulation of the latter. This suggest a
related to low oxygen and iron levels.
Using the techniques we applied for iron
restriction we could take a look at
qualitative as well as quantitative
changes of the cell wall proteome
depending on oxygen availability.
Not all proteins on the cell wall are
digested by trypsine or give peptides of
which MASCOT is able to use. These
proteins can be identified using
immunoblot assays. Sosinka et al.
(2008) have identified several cell wall
proteins using these assays at
6
vulvovaginal conditions under hypoxia
and iron restriction. It would be
interesting to identify these proteins as
well for pH 7.4.
W. LaJean Chaffin (2008). Candida
albicans
Cell
Wall
Proteins.
Microbiology and molecular biology
reviews, Sept. 2008, p. 495–544 Vol.
72, No. 3
Acknowledgement
I thank Stanley Brul and Frans Klis of
the Microbial Food Safety Group at the
Swammerdam Institute for Life Science
for giving the opportunity to help working
in a real running research. Special
thanks go out for Alice Sorgo and
Clemens J. Heilmann who have
supervised and helped during the
internship.
References
Daniel J. Kosman (2003). Molecular
mechanisms of iron uptake in fungi.
Molecular Microbiology, 47 (5), 1185–
1197
Robert Sutak, Emmanuel Lesuisse,
Jan Tachezy and Des R. Richardson.
Crusade for iron: iron uptake in
unicellular
eukaryotes
and
its
significance for virulence.
Ricardo S. Almeida, Sascha Brunke,
Antje Albrecht, Sascha Thewes,
Michael Laue, John E. Edwards, Jr.,
Scott G. Filler, Bernhard Hube (2008).
The Hyphal-Associated Adhesin and
Invasin Als3 of Candida albicans
Mediates Iron Acquisition from Host
Ferritin. PLoS Pathogen November
2008 ,Volume 4, Issue 11
Grazyna J. Sosinska, Piet W. J. de
Groot, M. Joost Teixeira de Mattos,
Henk L. Dekker, Chris G. de Koster,
Klaas J. Hellingwerf and Frans M.
Klis (2008). Hypoxic conditions and iron
restriction affect the cell-wall proteome
of Candida albicans grown under
vagina-simulative
conditions,
Microbiology, 154, 510–520
Mathias L. Richard and Armeˆl Plaine
(2007). Comprehensive Analysis of
Glycosylphosphatidylinositol-Anchored
Proteins in Candida albicans. Eukaryotic
Cell, p. 119–133 Vol. 6, No. 2
James G. Rohrbough, John N.
Galgiani and Vicki H. Wysocki (2007).
The
Application
of
Proteomic
Techniques
to
Fungal
Protein
Identification and Quantification. Ann.
N.Y. Acad. Sci. 1111: 133–146
Saif
Hameed,
Tulika
Prasad,
Dibyendu Banerjee, Aparna Chandra,
Chinmay K. Mukhopadhyay, Shyamal
K. Goswami, Ali Abdul Lattif, Jyotsna
Chandra, Pranab K. Mukherjee,
Mahmoud
A.
Ghannoum
and
Rajendra
Prasad
(2008).
Iron
deprivation inducesEFG1 -mediated
hyphal development in Candida albicans
without affecting biofilm formation,
Federation of European Microbiological
Societies FEMS Yeast Res 8 744–755
Tulika Prasad, Aparna Chandra,
Chinmay K. Mukhopadhyay and
Rajendra Prasad (2006). Unexpected
Link between Iron and Drug Resistance
of Candida spp.: Iron Depletion
Enhances Membrane Fluidity and Drug
Diffusion, Leading to Drug-Susceptible
Cells.
Antimicrobial
Agents
and
Chemotherapy, Nov. 2006, p. 3597–
3606 Vol. 50, No. 11
D.C. Sheppard, M. Zhang, A.S.
Ibrahim, Y. Fu, S.G. Filler, J.E.
Edwards (2003). A Functional Analysis
of the C. albicans Als Family of Proteins.
Interscience
Conference
on
Antimicrobial Agents and Chemotherapy
7
Janet F. Staab, Steven D. Bradway,
Paul L. Fidel and Paula Sundstrom
(1999). Adhesive and mammalian
transglutaminase substrate properties of
Candida albicans Hwp1. Science 283
1535-1538
Hong Xin, Sebastian Dziadek, David
R. Bundle, and Jim E. Cutler (2008).
Synthetic
glycopeptide
vaccines
combining –mannan and peptide
epitopes induce protection against
candidiasis. PNAS, vol. 105, nr 36
De Groot, P. W. J., De Boer, A. D.,
Cunningham, J., Dekker, H. L., De
Jong, L., Hellingwerf, K. J., De Koster,
C. & Klis, F. M. (2004). Proteomic
analysis of Candida albicans cell walls
reveals covalently bound carbohydrateactive enzymes and adhesins. Eukaryot
Cell 3, 955–965.
Qing Yuan Yin, Piet W.J. de Groot,
Chris G. de Koster and Frans M. Klis.
Mass spectrometry-based proteomics of
fungal wall glycoproteins.
8
Supplementary data.
Proteins
Als1
Als2
Als3
Nomenclature
Agglutinin-Like Sequence 1
Agglutinin-Like Sequence 2
Agglutinin-Like Sequence 3
Als4
Agglutinin-Like Sequence 4
Als5
Cht2
Crh11
Agglutinin-Like Sequence 5
Chitinase 2
Congo Red Hypersensitive 11
Ecm33
Mp65
ExtraCellular Mutant 1
MannoProtein of 65 kDa
Pga4
Phr1
Phr2
PH Responsive 1
PH Responsive 2
Pir1
Proteins with Internal Repeats 1
Rbt5
Rhd3
Sap9
Repressed By TUP1 5
Repressed during Hyphae
Development 3
Secreted Aspartyl Proteinase
Sod4
Sod5
Superoxide dismutase 4
Superoxide dismutase 5
Ssr1
Utr2
Cell-Surface Factor
Ywp1
Yeast-form Wall Protein
Function and features
Adhesion, role in virulence
Adhesion, biofilm formation
Epithelial adhesion,
endothelial invasiveness,
Iron assimilation
Adhesion, germ-tube
induction
Cell-cell adhesion
Chitinase
predicted glycosyl hydrolase
domain
Cell wall architecture
possible role in cell-wall
glucan metabolism
Cell wall organization
Glycosidase
Glycosidase
Structural constituent of cell
wall
hemoglobin utilization
Adhesion, cell surface
integrity
Superoxide dismutase
protective role against
oxidative stress
Cell wall structure
Putative glycosidase, cell
wall, adhesion
dispersal in host
Induction
Low iron
Hyphae
Down-regulated upon
vaginal contact
Yeast cells
fluconazole
heat
Oral candidiasis
High pH
Low pH, High Iron,
Fluconazole
Hyphal repressed,
fluconazole
High pH
Decrease in hyphae,
regulated by iron
fluconazole
Hyphal growth, osmotic and
oxidative stress
antifungals
cell wall regeneration
growth phase
Table S1. Descriptions of all proteins identified by MASCOT in a clean sample when using our cell wall isolation protocol
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