EUKARYOTIC CELL, Nov. 2007, p. 2056–2065
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 6, No. 11
Candida albicans Sun41p, a Putative Glycosidase, Is Involved in
Morphogenesis, Cell Wall Biogenesis, and Biofilm Formation?
Ekkehard Hiller,2Sonja Heine,1Herwig Brunner,1,2and Steffen Rupp1*
Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstrasse 12, 70569 Stuttgart, Germany,1and
Institute for Interfacial Engineering, University of Stuttgart, Nobelstrasse 12, 70569 Stuttgart, Germany2
Received 7 August 2007/Accepted 12 September 2007
The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of
proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different
cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans
the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking
SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41
mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was
observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a
defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae
on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of
cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as
a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation,
indicating an important role in the host-pathogen interaction.
Candida albicans is the most frequent causative agent of
candidiasis, which is among the most important nosocomial
infections of humans. As a commensal organism, present in the
gastrointestinal or urogenital tract of 30 to 60% of the popu-
lation, this opportunistic pathogen is widely spread and a con-
stant risk for immunocompromised patients (16). In recent
years, some key mechanisms and factors critical for virulence
in C. albicans were characterized, including polymorphic
switches and secreted proteases and lipases, as well as proteins
localized to the cell wall, like the adhesins (10, 11, 19). The cell
wall and its components are prime targets for antifungal strat-
egies, as they mediate the host-pathogen interaction. In addi-
tion, no similar structure exists in the host, facilitating targeted
development of antifungals. The SUN gene family comprises a
family of fungus-specific proteins which has been defined in
Saccharomyces cerevisiae as a group of four proteins, Sim1p,
Uth1p, Nca3p, and Sun4p, with highly similar C termini (3).
This gene family is involved in different cellular processes, like
DNA replication, aging, mitochondrial biogenesis, and cytoki-
nesis. Glucosidase activity has been assigned to this family of
proteins due to the characterization of the ?-glycosidase
BglBp, homologous to the SUN family in Candida wickerhamii
(32). In C. albicans, the SUN family comprises only two
genes—SUN41, the ortholog of SUN4, and SIM1, the ortholog
of UTH1—which have not been characterized in detail. SUN41
was previously found to be up-regulated in hyphae, as demon-
strated by Sohn et al., indicating a role in morphogenesis (33).
In addition, Sun41p has been mentioned as a putative sub-
strate for KEX2, a serine protease of the kexin superfamily,
which participates in both the constitutive and regulated se-
cretory pathways, indicating secretion (25). The ortholog
SUN4 in S. cerevisiae is involved in cell septation (21). The
corresponding protein, Sun4p, was identified as a glycosylated
soluble cell wall protein, extractable by reductive agents (4).
The association of Sun4p to the cell wall was also demon-
strated by Velour et al., who additionally were able to show a
localization of Sun4p to mitochondria (35). Furthermore, ge-
netic interactions of various degrees between the four SUN
family proteins have been reported for S. cerevisiae (21, 22).
We were interested in the involvement of Sun41p from C.
albicans in morphogenesis, cell wall biogenesis, and the host-
pathogen interaction. Therefore, we constructed SUN41 deletion
under various conditions.
Our results indicate that SUN41p has a function as a glyco-
sidase and is involved in morphogenesis and cell wall biogen-
esis as well as biofilm formation. SUN41 deletion mutants
failed to grow as hyphae on solidified media and showed a
decreased ability to adhere to these media. In addition, adher-
ence to tissue was also negatively affected in strains lacking
SUN41, as was the ability to form biofilms. Most interestingly,
Sun41p was found to be secreted from blastospores as well as
hyphae, pointing to the possibility of additional functions of
Sun41p in C. albicans, e.g., in the formation of extracellular
MATERIALS AND METHODS
Strains and growth conditions. C. albicans strains used in this study are listed
in Table 1. The strains were routinely inoculated from overnight YPD cultures
(10 g yeast extract, 20 g peptone, and 20 g glucose per liter) into fresh medium
and grown at 30°C on a rotary shaker for 6 h. Hyphae were induced in YPD plus
10% heat-inactivated fetal calf serum at 37°C or in ?-MEM plus 2% glucose at
37°C. Difco yeast nitrogen base (YNB; without amino acids; Becton Dickinson,
Heidelberg, Germany) supplemented with 75 mM ammonium sulfate and 2%
glucose was used as synthetic medium. Spider medium contained 1% nutrient
broth, 0.2% K2HPO4, and 1% mannitol as a carbon source (18). For growth on
plates, 1.5% agar was added to the medium.
* Corresponding author. Mailing address: Fraunhofer-IGB, Nobel-
strasse 12, 70569 Stuttgart, Germany. Phone: 49-711-970-4045. Fax:
49-711-970-4200. E-mail: Steffen.Rupp@igb.fhg.de.
?Published ahead of print on 28 September 2007.
Deletion of SUN41 and reconstitution of mutant strains. SUN41 was deleted in
C. albicans strain SC5314 by FLIP-mediated, site-specific recombination according
to the methods of Reuß et al. (29). Briefly, one pair of sequences flanking the
SUN41 coding sequence was amplified by PCR using the following primers:
3?) and orf6.2071_FLR1a_Xho1 (5?-CCGCTCGAGTGCTGGAAGTACTGCAC
ATAATTT-3?) for FLR1; orf6.2071_FLR2_Not1 (5?-ATAAGAATGCGGCCGC
ACCCCTTTTTCTCTTCTTCCTT-3?) and orf6.2071_FLR2_Sac2 (5?-TCCCCGC
GGACCGAAAAATCTTTGGCAGA-3?) for FLR2. Unique cleavage sites were
embedded in the primer sequences for directed ligation of the flanking regions into
the plasmid pSFS2A (29). FLR1 was cloned into the vector after ApaI and XhoI
digestion, and FLR2 was cloned after NotI and SacII digestion, resulting in the
plasmid pSFS2A-sun1a-2. Reconstituted mutants were produced in a similar way.
The SUN41 open reading frame, including 1,031 bp upstream, was PCR amplified
with the primers orf6.2071_FLR1a_Apa1 and 2071_end_rev_Xho1 (5?-CCGCTCG
AGGGAAGAAGAGAAAAAGGGGTATC-3?) and cloned together with FLR2
into pSFS1A (29) using the restriction sites ApaI/XhoI and NotI/SacII for FLR2. C.
albicans strains were transformed by electroporation as described previously (29).
Deletion and reconstitution were confirmed by Southern blotting and real-
Isolation of chromosomal DNA and Southern hybridization. Chromosomal
DNA from C. albicans cells was isolated as described according to the method of
Hoffman and Winston (9). Southern blot analysis was performed according to
standard protocols using 25 ?g of BclI-digested genomic DNA. To show the gene
deletion, the blots were probed with a DNA fragment that was PCR amplified
using primers for FLR2 (see above). The reconstitution was detected by a probe
amplified with SUN41_Rev_4 (5?-ACCGTTGTTGGAGGTGGTAG-3?) and
2071_end_rev_Xho1. After hybridization, Southern blots were exposed to phos-
phor screens (Fujifilm, Duesseldorf, Germany) for 24 h. The screens were
scanned using a FLA-5100 scanner (Fujifilm).
Growth rate determinations. The doubling times of the C. albicans strains
were determined by growing them in liquid YPD medium on a rotary shaker at
30°C. At 1-hour intervals, an aliquot was removed and sonicated briefly, followed
by measurement of the optical density at 600 nm (OD600).
Cell size determination. Cells were grown under standard conditions in YPD
and, after brief sonication, cells were collected by centrifugation. The cell pellet
was incubated in Brilliant Blue staining solution to contrast cells followed by two
washing steps in phosphate-buffered saline (PBS). Resuspended cells were im-
aged using light microscopy, and the contrast of cells to background was en-
hanced using Adobe Photoshop software. Afterward, the area of single cells was
determined with the software Scion Image (Scion Corporation). The software
Origin (OriginLab Corporation) was used to analyze the data. Two sample
independent t tests were performed.
Light microscopy. C. albicans strains were grown under standard conditions to
exponential phase and imaged using an Olympus BX 60 microscope (Olympus,
Scanning electron microscopy. C. albicans strains for scanning electron mi-
croscopy were grown under standard conditions to exponential phase. Cells were
washed two times with H2O and dropped onto glass slides. After freezing, cells
were dried by lyophilization and were sputtered according to standard protocols.
Scanning electron microscopy was performed using the 1530 VP electron micro-
Transmission electron microscopy. Samples for transmission electron micros-
copy were prepared according to the protocol described by Reinhard et al. (28).
Briefly, 3 ? 107blastospores of each strain were collected by centrifugation at
1,600 ? g after 6 h of growth in liquid YPD medium and fixed for 1 h by addition
of glutaraldehyde at a final concentration of 2.5%. The samples were then
washed with YPD and postfixed for 1 h with 1% osmium tetroxide at room
temperature. The fixed cells were dehydrated through a graded series of acetone
and embedded in Spurr’s resin (Sigma-Aldrich). Thin sections were stained with
uranyl acetate and lead citrate and then imaged with a Zeiss EM 10 electron
Plate assays. Cultures of C. albicans strains were inoculated from overnight
cultures, grown to an optical density at 600 nm of 1, and gently sonicated. Series
of 10-fold dilutions were prepared, and approximately 105, 104, 103, 102, and 101
cells were spotted onto YPD plates supplemented with 10% serum, YNB plates
with Congo red (300 ?g/ml), or ?-MEM plates. As a control, cells were applied
to YPD plates without any supplement. The plates were documented after
incubation at 30°C or 37°C for 5 days using a digital camera (Easyshare Z7590;
Kodak GmbH, Stuttgart, Germany) or a flatbed digital scanner (Epson Expres-
sion 1680 Pro).
Congo red. C. albicans strains were inoculated from overnight cultures and
grown to exponential phase in liquid YPD medium which was supplemented with
Congo red (300 ?g/ml).
Wash test. C. albicans strains were inoculated from overnight YPD plates to
fresh YPD plates (with or without 10% fetal calf serum). After growth for 48 h
(30°C or 37°C for hyphae), cell films were washed for 2 min with double-distilled
H2O on a circular shaker at 75 rpm. The same procedure was used for plates
containing additional Congo red (300 ?g/ml).
Glucanase treatment. Cells were treated with the recombinant ?-1,3-glucanase
Quantazyme ylg (MP Biomedicals, Illkirch, France) according to the protocol of
the manufacturer. The OD600was measured before and every minute after
addition of 500 U/ml enzyme. Data were plotted against time, and the time
required for a 50% decrease in the OD was determined as the half-life for
Cell culture. The human colorectal adenocarcinoma cell line Caco-2 (ATCC
HTB-37) was grown in 75 cm2tissue culture flasks (Greiner Bio-One, Fricken-
hausen, Germany). Dulbecco’s modified Eagle’s medium (Gibco) supplemented
with 10% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, and 1%
gentamicin was used as the medium. Cells were cultivated at 37°C under 5%
CO2. Just before reaching confluence, Caco-2 cell cultures were split 1:5 by
Adhesion assay. Twenty-four-well polystyrene plates were employed to study
the adhesion behavior of C. albicans on different surfaces as described previously
(5). The quantitative data were based on four experiments.
Biofilm formation. Biofilms from different C. albicans strains were produced
on sterilized, polystyrene, flat-bottom 96-well microtiter plates (Greiner Bio-
One) as described previously (12) with some modifications. Briefly, 100 ?l of a
standardized cell suspension (3 ? 107cells/ml) was transferred into each well
(eight replicas) of a microtiter plate and incubated for 1.5 h at 37°C to allow the
yeast to adhere to the surfaces of the wells (15). As controls, eight wells of the
microtiter plate were handled in an identical fashion, except that no Candida
suspensions were added. Following the adhesion phase, the cell suspensions were
aspirated and each well was washed twice with 150 ?l of PBS to remove loosely
adherent cells. A total of 100 ?l of yeast nitrogen base medium was then
transferred into each of the wells, and the plates were incubated at 37°C. The
biofilms were allowed to develop for 48 h and then the yeasts were quantified
by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) reduction assay (see below).
XTT reduction assay. The XTT reduction assay used was a modification of the
methods described previously (15). Briefly, XTT (Sigma-Aldrich) solution (1
mg/ml in PBS) and menadione (Sigma) solution (1 M in acetone) were prepared
immediately before each assay. XTT solution was mixed with the menadione
solution at a ratio of 1,000:1 by volume. The biofilms were first washed two times
with 200 ?l of PBS, and then 100 ?l of the XTT-menadione solution was added
to each well. The plate was then incubated in the dark for 2 h at 37°C. Following
TABLE 1. C. albicans strains
Strain name(s)Description Genotypea
Can417 and Can423
Can418 and Can424
Can419 and Can425
Can420 and Can426
Can421 and Can427
Can422 and Can428
Precursor of sun41?
Precursor of sun41?
Precursor of sun41?
Precursor of sun41?-SUN41
aFRT, FLP recombinase target (20).
VOL. 6, 2007 Sun41p IS INVOLVED IN MORPHOGENESIS AND ADHESION2057
incubation, 80-?l aliquots of solution were transferred to new wells and the color
change in the solution was measured at 490 nm with a microtiter plate reader
(SpectraMax Plus microplate spectrophotometer; Molecular Devices, Ltd.,
Sunnyvale, CA). The absorbance values of the controls were then subtracted
from the values of the test wells to eliminate spurious results due to background
RNA preparation. C. albicans cells were grown in suspension cultures as
described before. Cells were collected by centrifugation at 1,600 ? g and dropped
into liquid nitrogen for generating cell pearls. These cell pearls were stored at
?80°C. In order to isolate total RNA, the cell pearls were broken mechanically
by grinding in a precooled Retsch mill (Retsch, Haan, Germany), and RNA was
further purified using the RNeasy mini kit (Qiagen, Hilden, Germany), following
the instructions of the manufacturer. The quality of the isolated RNA was
checked by agarose electrophoresis prior to analysis.
Real-time PCR. A selection of all enzymes annotated as modifying the major
backbone of the cell wall was tested. Amounts of cDNA of selected genes in the
sun41? mutant strain were compared to the wild type by using TDH3 (glyceral-
dehyde-3-phosphate dehydrogenase) as a reference and assuming an equal
amount of transcript of this gene in the samples. Additionally, the transcript
amounts of the genes of interest were compared to the amount of TDH3 tran-
script to get an idea about the abundance of the respective genes. One ?g total
RNA was transcribed to cDNA using the QuantiTec reverse transcription kit
(Qiagen). Two ng of this cDNA was used to perform real-time PCR in a
LightCycler 480 (Roche, Mannheim, Germany) with the LightCycler 480 probes
master kit according to the manufacturer’s instructions on 96-multiwell plates.
Probes used (as indicated) originated from the human Universal ProbeLibrary
(Roche), and gene-specific primers (TIB MOLBIO, Berlin, Germany) were as
follows: for BGL2 (orf19.4565), 5?-TGAAGCTGAAAAGGAAGCTTTG-3?, 5?-
GCTTCAGAACCAACCAAGAAA-3?, and probe 22; for PHR1 (orf19.3829),
and probe 6; for ACF2 (orf19.3417), 5?-TCAAACATCAGCAACAACCAA-3?,
5?-TGTATGGATTGGGGCATCTT-3?, and probe 89; for ENG1 (orf19.3066),
3?, and probe 18; for SCW11 (orf19.3893), 5?-ACCACACAATCACCTTCCAC
T-3?, 5?-TTGGACTAGAGGTTGAGGTTGAG-3?, and probe 11; for XOG1
(orf19.2990), 5?-TGCTAAATGGTTGAATGGTGTC-3?, 5?-GCATTATCGTA
AGCACCCTCA-3?, and probe 82; for ACE2 (orf19.6124), 5?-TCATCGCCAG
AACCACATT-3?, 5?-GGATATCTGTTGCGGTGGTT-3?, and probe 60; for
SIM1 (orf19.5032), 5?-CCAGTTGTTTTGGGATCTGG-3?, 5?-TGGGTTTGGA
ATCAATGACA-3?, and probe 131; for CBK1 (orf19.4909), 5?-AACCACAAC
AGCAGCAACAA-3?, 5?-GCTGCTGCTGGAATATTTGC-3?, and probe 5; for
CHT3 (orf19.7586), 5?-GGTGCTGCTGGATCTTATGG-3?, 5?-CCCAAAGAG
TATGAGCAAATTGT-3?, and probe 59; for SUN41 (orf19.3642), 5?-CAGGT
GA-3?, and probe 10; for PHR2 (orf19.6081), 5?-GTTTCATTAGCCGACTAC
TTTGC-3?, 5?-TGTTTATACCGAAAAAGTCAGCAG-3?, and probe 9; for
TDH3 (orf19.6814), 5?-GCCGTCAACGATCCATTC-3?, 5?-AGAATCGTATT
TGAACATGTAAGCA-3?, and probe 50. The LC480 software (Roche) was
used to analyze the data.
Isolation of secreted proteins. C. albicans was inoculated after two washing
steps in H2O in fresh synthetic medium YNB or ?-MEM and grown for 7 h. Cells
were removed from 10 ml of medium by three rounds of centrifugation (3,500 ?
g, 3,500 ? g, and 12,000 ? g). Proteins were precipitated out of the medium by
methanol-chloroform (36). Protein pellets were dissolved in 100 ?l 100 mM
ammonium bicarbonate. Reduction and alkylation of disulfide chains were
achieved by addition of 10 mM dithiothreitol and incubation at 65°C for 15 min,
followed by adding iodoacetamide to a final concentration of 20 mM and incu-
bation at room temperature in the dark for 15 min. Thereupon, 1 ?g trypsin
(sequencing grade, modified; Promega, Mannheim, Germany) was added, and
the digest mixture was incubated for 5 h at 37°C before it was stopped by adding
trifluoroacetic acid to a final concentration of 1%. Analysis of peptides was
performed by tandem mass spectrometry (MS/MS) using matrix-assisted laser
desorption ionization–time-of-flight (MALDI-TOF) or liquid chromatography-
coupled electrospray ionization (ESI).
MALDI-TOF MS. C. albicans was grown in the synthetic medium YNB or
?-MEM for 7 h before collecting the supernatant. After precipitation of the
proteins, they were resuspended and digested using trypsin. Peptides in a volume
of 10 ?l were purified by ZipTipC18(Millipore, Billerica, MA) using a standard
protocol and eluted in 1 ?l directly on a prespotted AnchorChip target (Bruker
Daltonik GmbH, Bremen, Germany). The monoisotopic molecular mass of the
peptides was analyzed by MALDI-TOF MS using an Ultraflex II TOF/TOF 200
apparatus (Bruker). The mass spectrometer was set to scan over 700 to 4,000 Da,
and the mass spectra were acquired in reflector mode and processed using
FlexControl and FlexAnalysis software (Bruker). The most dominant peaks were
further microsequenced using post-source decay analysis.
LC-MS/MS. Reverse-phase liquid chromatography (LC)–MS/MS was per-
formed using a Surveyor LC system (Thermo Fisher Scientific, Ulm, Germany)
which was coupled to a Finnigan LCQDECAmass spectrometer (Thermo)
equipped with an electrospray ion source.
The peptide mixtures were autosampled in 0.1% aqueous trifluoroacetic acid
and separated on the analytical column (Jupiter; C18, 1 mm [inner diameter] by
10 cm; Phenomenex) using a linear gradient of 7 to 100% acetonitrile in 0.1%
(vol/vol) formic acid for 44 min at a flow rate of 50 ?l/min and ionized by an
applied voltage of 5 kV to the emitter. The mass spectrometer was operated in
data-dependent acquisition mode to automatically switch between MS and MS/
MS. Survey MS spectra were acquired for 1 s, and the most intense ions were
isolated and sequentially fragmented for 1.5 s by low-energy collision-induced
dissociation. The mass spectrometer was set to scan over 300 to 2,000 Da, and the
mass spectra were acquired and processed using Xcalibur software (Thermo).
Analysis of mass spectrometric data. The tandem MS spectra were submitted
to the database search program MASCOT (Matrix Science, Great Britain) in
order to identify the proteins. Data files were searched against a C. albicans
database. This database is based on assembly 19 of the translated open reading
frames of the nucleotide sequence of the Stanford Genome Technology Center.
The MASCOT search parameters were the following: allowing up to one missed
cleavage, a tolerance of 1.5 Da for peptides and 1 Da for MS/MS (MALDI-
TOF/TOF, 100 ppm for peptides and 0.5 Da for MS/MS). Probability-based
MASCOT scores were used to evaluate protein identifications. Only peptides
with P values of ?0.05 for random occurrence were considered to be significant.
The switch from yeast to hyphae has been shown to be
crucial for virulence of C. albicans. Genes showing up-regu-
lated expression during this step are of special interest to
understanding pathogenicity. In a study by Sohn et al. (33),
SUN41 was found to be up-regulated during hyphal develop-
ment. For further functional characterization, we constructed
sun41? mutant strains by transformation and homologous re-
combination with the SAT1 flipper cassette (29). SUN41 was
shown not to be essential in C. albicans. Homozygous sun41?
mutants were reverted with the SUN41 gene under its native
promoter (Table 1). Southern blot analyses confirmed the ge-
notype, and real-time PCR confirmed the absence of SUN41
transcription in the homozygous sun41? mutant strains (not
Phenotypic analysis of sun41? cells. (i) Deletion of SUN41
results in increased cell size and cytokinesis defects. Deletion
of SUN41 did not affect growth rate during growth in the
exponential phase in YPD (not shown). However, an increased
flocculation could be observed in liquid cultures of the sun41?
mutant compared to the wild type. This occurred during
growth as blastospores in YPD medium (Fig. 1) as well as for
hyphae developing in ?-MEM at 37°C (not shown). Light mi-
croscopy of exponentially growing cells revealed that the
sun41? blastospores appeared irregular, not separated after
budding, in a tree-like structure (Fig. 1B). This phenotype was
stable even after gentle sonication, which separates cells just
sticking together. To get a more detailed view of the cells and
the nonseparated budding sites, electron microscopy was per-
formed. Scanning electron microscopy confirmed that sun41?
blastospores were still connected at the site of the bud/birth
scar, even after completion of budding, indicated by the start of
the next cell cycle of former mother and daughter cells (Fig.
1D and E). Using transmission electron microscopy to inves-
tigate the bud sites inside of the cells, we did not observe any
significant differences in cell wall appearance between separat-
2058HILLER ET AL.EUKARYOT. CELL
ing wild-type cells (Fig. 1F) and still-connected sun41? mutant
cells (Fig. 1G). A thin contrasted line at the sun41? mutant’s
budding site seemed to indicate the completion of the cell walls
and septae from both sides, just missing the final cut (Fig. 1H).
SUN4 deletion mutants of S. cerevisiae, the orthologous gene
to C. albicans SUN41, showed an increase in cell size. We
investigated this phenotype also in C. albicans. Measurement
of the cell size on light micrographs of ultrasonically separated
cells showed that sun41? mutants were enlarged during expo-
nential growth at about 1.3 times that of the wild type (Fig. 2).
This effect was reduced in the reconstituted strain sun41?-
SUN41. The reason for this cell size enlargement remains
sun41? mutant cells grown in liquid culture under hyphae-
inducing conditions, like ?-MEM or YPD supplemented with
10% bovine calf serum at 37°C, revealed no difference in mor-
phology compared to wild type. Only a slightly reduced acidifica-
tion of the ?-MEM and enhanced flocculation, as mentioned
above, could be observed (not shown).
(ii) sun41? mutants show altered sensitivity to cell wall-
modifying substances. The sensitivity towards cell wall-per-
turbing substances was tested by a survival test on plates con-
taining Congo red or calcofluor white. Congo red interferes
with the assembly of the glucan microfibrils (14), and cal-
cofluor white disturbs chitin assembly (6). Mutant and wild-
type cells were diluted in a serial manner and grown for up to
5 days on supplemented agar plates. sun41? cells showed a
higher sensitivity towards Congo red, whereas no effect with
calcofluor white was observed (Fig. 3B and data not shown).
This indicates a variation in the glucan network, possibly re-
sulting in a defect in cell wall structure. Therefore, the sensi-
tivity of the cells against the cell wall-corrupting ?-1,3-glu-
canase Quantazyme was analyzed. Cells harvested during
exponential growth were tested for the half-life of spheroplast
lysis (time required for a 50% decrease in the OD). sun41?
mutants (42-min half-life) showed more resistance towards
?-1,3-glucanase activity than the wild type (21-min half-life) or
reconstituted sun41?-SUN41 (28-min half-life), indicating
compensatory mechanisms to strengthen the cell wall possibly
in combination with a reduced amount of ?-1,3 linkages in the
Using calcofluor white in fluorescence microscopy to stain
chitin of growing cells, no differences between the wild type
and sun41? mutants were observed on blastospores and hy-
phae (not shown), indicating no severe changes in chitin struc-
Most interestingly, C. albicans wild-type strains incubated in
liquid YPD medium supplemented with Congo red also
showed an increased flocculation. Light microscopy of expo-
nentially growing cells revealed that the blastospores did not
FIG. 1. Microscopic imaging of different C. albicans strains growing
exponentially in liquid YPD. After 6 h, cells were fixed with glutaral-
dehyde and imaged using light microscopy (A and B) or prepared for
further imaging with either scanning electron microscopy (C to E) or
transmission electron microscopy (F to H). Bars, 2 ?m.
FIG. 2. Distribution of cell sizes of different C. albicans strains. The
sizes of the cell surfaces of C. albicans strains on microscopic images
were measured and plotted on a box-whisker plot. The distribution
differed significantly between the wild type and ?sun41 mutant (P ?
7.2 ? 10?14). The boxes indicate the interquartile ranges (25th to 75th
percentiles), the horizontal thick lines symbolize the medians, the
black squares represent the means, the whiskers extend to 1.5 times the
interquartile range, and the circles illustrate the outliers.
FIG. 3. Influence of Congo red on different C. albicans strains.
Serial dilutions of C. albicans cells of the indicated strains were spotted
onto agar plates containing YPD (A) or YNB (B) supplemented with
300 ?g/ml Congo red. Plates were incubated for 5 days at 30°C. (C) C.
albicans wild-type cells were incubated in the indicated media at 30°C
for 7 h. (D) C. albicans wild-type and sun41? cells were incubated in
YPD supplemented with 10% serum and 300 ?g/ml Congo red at 37°C
for 7 h. Bars, 10 ?m.
VOL. 6, 2007 Sun41p IS INVOLVED IN MORPHOGENESIS AND ADHESION2059
separate after budding, growing in a tree-like structure, similar
to the phenotype observed with sun41? (Fig. 3C).
(iii) SUN41 is required for hypha formation on solid me-
dium. Formation of hyphae is important for C. albicans to
invade into the host tissue. In liquid medium under hypha-
inducing conditions, sun41? mutants did not show differences
in hyphae formation compared to the wild type (see above).
Investigation of filamentation during growth on solid medium
revealed a remarkable difference from this result. Exponen-
tially grown C. albicans cells were diluted and cultivated on
agar plates containing ?-MEM, YPD with 10% bovine calf
serum, or Spider medium for up to 5 days at 37°C. Whereas the
wild type and reconstituted sun41?-SUN41 showed peripheral
hyphae and hyphal cells within the colony, this switch in growth
form was barely detectable in the sun41? mutant, revealing an
involvement of SUN41 in hypha formation on solidified me-
dium (Fig. 4A and B). Most interestingly, addition of Congo
red to sun41? strains in liquid medium (YPD plus serum,
37°C) blocked hyphae formation almost completely and re-
sulted again in the formation of blastospore-like cells not sep-
arated from each other, as observed on solidified medium or in
liquid YPD at 30°C. The hyphae formation of the wild type was
affected only moderately by Congo red under the conditions
tested (Fig. 3D).
(iv) sun41? shows defects in adhesion and reduced biofilm
formation. Adhesion of sun41? cells was tested by several
different assays: on agar plates, using an epithelial model of
host-pathogen interaction, and in a biofilm assay. On agar
plates, depending on the medium used, several distinct pheno-
types were observed. On YPD plates after 24 h of growth,
sun41? strains adhered more strongly than the wild type and
the reconstituted strain in a plate washing assay (Fig. 4C).
Microscopic observation of plate-grown sun41? strains re-
vealed the same phenotype of mutant cells, a cytokinesis de-
fect, as described for liquid YPD medium (see above). The
increased adhesion observed may be due to the increased cell-
cell interaction in the mutant strain as a result of the cytoki-
nesis defect. To test whether the separation defect is the cause
of increased adherence on agar plates, we incubated the wild
type and sun41? mutant and its reconstituted strain for 48 h on
YPD plates containing Congo red, which induces a cytokinesis
defect in these strains, as described above. Consistent with the
idea that the defect in cytokinesis is responsible for the in-
creased adherence, an equal amount of cells from all strains
persisted on the plates after washing (data not shown). Micros-
copy confirmed that cells of all strains showed the tree-like
structure on solid media as observed in liquid media supple-
mented with Congo red (Fig. 3C).
On hypha-inducing media, however, sun41? colonies were
found to be washed away much easier from the plates than
hyphal wild-type cells, indicating that the tree-like structures
also observed on hypha-inducing solid medium with sun41?
mutant cells have a reduced potential to adhere and invade in
the agar than hyphal filaments present in the wild type (Fig.
Adhesion of C. albicans on epithelia is a necessary precon-
dition for penetration of tissues in order to mediate pathogen-
esis in the host. To test the differences in adhesion of wild-type
and sun41? strains, we used an in vitro adhesion assay (5). For
this purpose, Candida cells were incubated to adhere to a
Caco-2 cell monolayer. To determine whether adhesion of
sun41? and the wild-type C. albicans strain occurs with differ-
ent kinetics or efficiencies, the numbers of C. albicans cells
adhering to the surface and those remaining in the supernatant
were analyzed in a time-dependent manner (Fig. 5A). Adhe-
sion of the wild type to Caco-2 cells was rapid and efficient, as
indicated by an adherence of 67% after the first 30 min. A
maximum of 93% was nearly reached after 90 min. After 30
min, 54% of sun41? C. albicans cells were found adhered to
the surface, reaching their maximum of 81% after 120 min. In
reconstituted sun41?-SUN41 cells, the same kinetics and effi-
ciency of adhesion as the wild type were observed (Fig. 5A).
Adhesion of sun41? to the plastic surface did not differ signif-
icantly from the wild type (not shown).
FIG. 4. Phenotype of different C. albicans strains growing on solid-
ified media. (A) Approximately 100 cells of the indicated C. albicans
strains were spotted onto agar plates containing ?-MEM or YPD
supplemented with 10% bovine calf serum. Plates were incubated for
5 days at 37°C. (B) The rim of the colonies grown on YPD supple-
mented with 10% bovine calf serum was imaged using light microscopy
(upper three panels). Additionally, cells were scraped off the colonies
and examined microscopically (lower three panels). (C) The ability to
adhere on YPD agar was tested by washing layers formed by the
indicated strains on plates with or without serum. Bars, 10 ?m.
2060HILLER ET AL.EUKARYOT. CELL
Both filamentation and adhesion of Candida are involved in
the formation of biofilms. These biofilms are found on medical
devices, like catheter surfaces (2), and have attracted attention
because of their persistence and resistance to antifungals (13,
31). These characteristics of Candida biofilms are likely to
contribute to both superficial (30) and systemic (26) candidi-
asis. We used an in vitro assay to investigate if the ability of the
sun41? C. albicans to form biofilms was altered. Cells were
added to 96-well plates and incubated until the cells adhered to
the surface. After removal of nonadhered cells, plates were
incubated for 2 days to allow development of a biofilm (12).
Equal amounts of cells were found of wild type and sun41?-
SUN41; sun41?, however, was found in much lower amounts
Deletion of SUN41 results in compensatory regulation in
other glycosidases. Real-time PCR was applied to study the
differential regulation on the transcriptional level and the
amount of transcript of selected genes between wild type and
sun41?. Testing ACE2, ACF2, BGL2, CBK1, CHT3, ENG1,
PHR1, PHR2, SCW11, SIM1, and XOG1 during growth as
blastospores, none of these genes was regulated to a factor less
than 0.5 or more than 2.0, indicating that the loss of SUN41
had no effect to the regulation of these genes (not shown). This
result is in contrast to hyphae, where we observed that PHR2
and ACF2 were up-regulated with a factor of ?2.0, and the
amount of SCW11 and CHT3 transcript in the sun41? mutant
was less than half that of the wild type (Fig. 6). Also, ACE2,
BGL2, and PHR1 seemed to be down-regulated but did not
reach the 0.5? regulation factor. Determination of the tran-
scription level revealed mostly a very low abundance, except
for BGL2, PHR1, PHR2, and SUN41.
Localization of Sun41p. Our observation of the phenotypic
effects of SUN41 deletion on blastospores indicated a function
during the last step of cell division. In this context, it is re-
markable that the Sun41p ortholog, Sun4p, in S. cerevisiae was
found in the cell wall (4). Attempts to localize Sun41p using a
C-terminal green fluorescent protein tag were not successful.
As shown by Lee et al., the amino acid sequence of SUN41
contains with high probability an N-terminal secretory signal
peptide, indicating entry in the secretory pathway (17; http:
//info.med.yale.edu/intmed/infdis/candida/). To test if Sun41p
is secreted, we used mass spectrometry. Comparison of the
tryptic peptide spectra of proteins in the supernatant of YNB
medium gained from wild-type and sun41? C. albicans cells
showed that very similar spectra could be observed. However,
the sun41? MALDI-TOF spectrum lacked two dominant
peaks of 1,048.629 Da and 3,794.181 Da (Fig. 7, peaks 1 and 5).
Sequencing by analysis of post-source decay spectra showed
that these peptides belong to Sun41p. Dominant peptides
found in both spectra belonging to Sim1p, MP65p, and Tos1p
were identified in an equal manner (Fig. 7, peaks 2, 3, and 4).
To verify these results, the tryptic digests were analyzed
in reverse-phase high-performance liquid chromatography
(HPLC)-coupled ESI MS/MS. This resulted in the repeated
identification of Sun41p in YNB as well as in ?-MEM obtained
from wild-type cells but not from sun41?. This indicates that
FIG. 5. (A) Quantitative assay to determine adherence of different
C. albicans strains on a Caco-2 monolayer. C. albicans strains were
applied to a monolayer of Caco-2 cells. The percentage of adherent C.
albicans cells after 30, 60, 120, and 240 min of infection is shown.
Results are average values with standard deviations of four indepen-
dent experiments. (B) Comparison of biofilm formation of different C.
albicans strains. C. albicans cells of the indicated strains were incu-
bated in 96-well plates for 2 days. The amount of biofilm-forming cells
was measured in an XTT activity assay. Results represent means with
standard deviations (error bars) from two independent experiments.
FIG. 6. Differential regulation in sun41?: total RNA of C. albicans
wild-type and sun41? mutant cells growing as hyphae in liquid ?-MEM
were collected and reverse transcribed to cDNA, which was used as a
template in a real-time PCR. The transcript amount of the indicated
genes in the sun41? mutant was compared to that of the wild type.
Dashed lines represent two times and one-half the amount of tran-
script in the sun41? mutant. The dotted line in the middle indicates an
equivalent amount (1.0?). Error bars indicate the standard errors of
the means from three independent experiments.
VOL. 6, 2007Sun41p IS INVOLVED IN MORPHOGENESIS AND ADHESION 2061
Sun41p, at least partially, is secreted under these conditions.
Several other proteins could be identified during this analysis
as being secreted into the medium (Table 2). With the excep-
tion of Dsl1p (Table 2), the proteins contain a typical signal
peptide characteristic for secreted proteins (17).
Following the detection of Sun41p in the supernatant, we
tested whether the secreted protein could compensate for the
blastospore separation defect of the sun41? mutant cells by
cultivating this strain together with the wild type. During in-
cubation for 24 h, numbers of mutant and wild-type cells were
determined. No significant change in separation of sun41?
cells was observed at any time point investigated (not shown).
We have investigated the role of SUN41 in cell wall biogen-
esis and morphogenesis in C. albicans. SUN41 of C. albicans
was annotated as homologous to the SUN4 gene in S. cerevi-
siae, sharing 57% identity and 70% similarity. In S. cerevisiae,
several functions have been assigned to SUN4, including cell
wall biogenesis, cell size, cytokinesis, and mitochondrial func-
tion (4, 35). In C. albicans our previous work identified SUN41
as transcriptionally up-regulated in hyphae (33). To identify
the functions of SUN41 in C. albicans, we constructed strains
deleted for SUN41 and compared them to the wild type under
different conditions. Homozygous deletion mutants exhibited
normal growth rates under the conditions tested, showing that
SUN41 is not essential. However, SUN41 mutant strains
showed several abnormalities with respect to cell wall biogen-
esis and morphogenesis. This included defects in cytokinesis,
biofilm formation, and hyphae formation on solid substrates as
well as defects in adhesion. In addition, the cell wall biogene-
sis-perturbing agent Congo red, but not calcofluor white, re-
sulted in reduced survival of strains lacking Sun41p.
Morphogenesis and cell wall biogenesis. Microscopic obser-
vations of sun41? blastospores revealed a cytokinesis defect
producing a tree-like structure. Furthermore, our results indi-
cate that sun41? blastospores are slightly larger than the wild
type. However, this phenotype is not restored completely in the
reconstituted strain, allowing other interpretations for this ef-
fect. A cytokinesis defect and cell enlargement have also been
reported for S. cerevisiae sun4? (21). Cells were not able to
separate after completion of cell division (Fig. 1B and D).
However, electron microscopy pictures revealed an intact cell
wall and septum similar to wild-type cells (Fig. 1G). More-
detailed electron micrographs focusing on differences at the
budding site might reveal potential structural changes leading
to this defect. The tree-like structure was observed for sun41?
mutant cells growing as blastospores in liquid media as well as
on solid media.
Most interestingly, we found that the deletion of SUN41 did
not affect hypha formation in liquid media like YPD plus
serum (37°C) or ?-MEM (37°C). However, on the respective
solid media, sun41? mutant strains did not form hyphae, but a
tree-like structure was observed for growth in liquid YPD
media, mixed with single or budding cells. Hyphae/pseudohy-
phae-like structures were barely observed, and true hyphae
could not be detected microscopically (Fig. 4B, middle lower
panel). This shows that sun41? mutant cells in general are able
to form hyphae; however, the morphogenetic program re-
quired to induce hyphae formation seems to be perturbed in
FIG. 7. Comparison of proteins found in supernatants of different C. albicans strains growing in YNB. Different C. albicans strains were grown
exponentially in the liquid synthetic medium YNB. Cells were removed and media collected. Proteins were precipitated and digested with trypsin.
Shown are the mass spectra of the resulting peptide mixtures from 700 to 4,000 Da from the MALDI-TOF MS. The sequences of the indicated
peptides were identified by MS/MS and associated to proteins using the MASCOT software. Peaks: 1 and 5, Sun41p; 2, Sim1p; 3, MP65p; 4, Tos1p.
2062HILLER ET AL.EUKARYOT. CELL
sun41? strains on solidified media. Comparing transcript levels
of several putative glycosidases between the wild type and
sun41? revealed a prominent up-regulation (factor of ?2) of
PHR2 in the deletion mutant if grown in liquid medium under
hypha-inducing conditions (MEM, 37°C) (Fig. 6). Phr2p has
been reported as a putative ?-1,3-glycosidase (7, 23). The com-
pensatory up-regulation of Phr2p, at pH 7.4, in a sun41? strain
would be consistent with a function of Sun41p as a ?-1,3-
glycosidase. This up-regulation was not found in blastospores,
where the cytokinesis defect was observed. In blastospores we
observed a higher resistance to ?-1,3-glucanase in sun41?
strains, which is in agreement with a function for Sun41p in cell
wall biogenesis, potentially as a ?-1,3-glycosidase.
Although sun41? strains are more resistant to ?-1,3-glu-
canase, they show a general defect in cell wall biogenesis as
observed by incubation on Congo red but not calcofluor
white plates (Fig. 3B and data not shown). This is also in
agreement with a putative function as a glycosidase, since
Congo red has been shown to interfere with glucan in the S.
cerevisiae cell wall and affects the assembly of the glucan
microfibril biogenesis (14, 34). Interestingly, the addition of
Congo red also resulted in a separation defect of C. albicans
wild-type blastospores, similar to the phenotype observed
for SUN41 deletion mutants (compare Fig. 1B to 3C). This
effect of Congo red was also observed in S. cerevisiae (14,
34). In hypha-inducing liquid medium, Congo red only had
a limited effect on the morphogenesis of the wild type;
however, sun41? strains predominantly form a tree-like
structure, as observed on the solid media tested. This indi-
cates that the potential compensatory effect observed by
induction of additional glycosidases in sun41? strains is
blocked by Congo red. These results suggest that cell wall
composition/alterations are sensed by C. albicans, resulting
in different decisions for morphogenesis.
TABLE 2. Proteins identified by MS/MS (MALDI-TOF and ESI) in media supernatants of wild-type C. albicans cells
growing in ?-MEM or YNB
Grown in ?-MEM
orf19.1779 Mp65p Yes
orf19.2370 Dsl1p No
Grown in YNB
orf19.1690 Tos1pYes EAGSVATIGY
aNumber of the open reading frame (ORF) according to assembly 19.
bPredicted by Lee et al. (17).
cSequences were identified using ESI MS/MS except for sequences with an asterisk, which were identified by MALDI-TOF MS/MS.
dExpectation value for the peptide match; values below 0.05 indicate identity or extensive homology.
VOL. 6, 2007Sun41p IS INVOLVED IN MORPHOGENESIS AND ADHESION2063
Adhesion and biofilm formation. The observed phenotypes
of sun41? strains also have significant impact on their ad-
hesive behavior. The inability to form hyphae on solid me-
dium results in strongly reduced adhesion compared to the
reconstituted strain or the wild type (Fig. 4C). A reduced
adhesion of sun41? cells was also observed on an epithelial
model consisting of Caco-2 cells (Fig. 5A). However, among
cells on YPD plates grown for 24 h, the cytokinesis defect
resulting in a tree-like structure of the mutant cells led to a
stronger adhesion of sun41? cells (Fig. 4C). By adding
Congo red to the YPD plates, both the wild type and the
mutant now adhered equally strongly, presumably due to the
induced cytokinesis defect (tree-like structure) present now
in all strains (Fig. 3C and data not shown). The degree of
adhesion observed on agar medium therefore directly re-
flects the degree of cellular networks, from single blasto-
spores to cells adherent due to a defect in cytokinesis all the
way to hyphae as a continuous network of cells.
We also observed a strong defect in biofilm formation (Fig.
5B). According to Nobile et al., three basic stages are necessary
for biofilm formation in vitro: (i) attachment of yeast cells to
the surface and (ii) growth and proliferation of yeast cells to
form a basal layer, followed by (iii) extensive filamentation
combined with the production of extracellular matrix (27).
Attachment of blastospores to a plastic surface was not signif-
icantly affected by deletion of SUN41 (data not shown). There-
fore, an inability to form hyphae on a solid support may be the
main reason for the observed defect in biofilm formation in
sun41? cells. In recent years, analysis of the biofilm matrix of
C. albicans revealed glucan as a major component (1, 24). This
indicates that the putative glycosidase Sun41p may have addi-
tional functions in the generation of the extracellular matrix in
biofilms. This hypothesis is supported by our observation that
Sun41p is a protein secreted into the medium (Fig. 7 and Table
2). We could not detect any effect of conditioned medium
containing Sun41p on sun41? strains with regard to cytokinesis
(data not shown), indicating potential additional functions of
the secreted Sun41p. Further studies will have to verify this
We thank Michael Schweikert for helpful assistance during trans-
mission electron microscopic imaging and Monika Riedl for perform-
ing the scanning electron microscopy. Also, we thank Martin Zavrel
for help with the cell culture assays. Joachim Morschha ¨user and Oliver
Reuß are acknowledged for the gift of plasmids. Sequence data were
obtained by the Stanford Technology Center at http://www-sequence
This work was supported by DFG Ru608/4-2.
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