Role of Bcr1-Activated Genes Hwp1 and Hyr1 in Candida
Albicans Oral Mucosal Biofilms and Neutrophil Evasion
Prabhat Dwivedi2, Angela Thompson1, Zhihong Xie1, Helena Kashleva1, Shantanu Ganguly3, Aaron P.
Mitchell3, Anna Dongari-Bagtzoglou1*
1Division of Periodontology, School of Dental Medicine, University of Connecticut, Farmington, Connecticut, United States of America, 2Department of Microbiology,
University of Texas, Houston, Texas, United States of America, 3Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of
Candida albicans triggers recurrent infections of the oropharyngeal mucosa that result from biofilm growth. Prior studies
have indicated that the transcription factor Bcr1 regulates biofilm formation in a catheter model, both in vitro and in vivo.
We thus hypothesized that Bcr1 plays similar roles in the formation of oral mucosal biofilms and tested this hypothesis in a
mouse model of oral infection. We found that a bcr1/bcr1 mutant did not form significant biofilm on the tongues of
immunocompromised mice, in contrast to reference and reconstituted strains that formed pseudomembranes covering
most of the tongue dorsal surface. Overexpression of HWP1, which specifies an epithelial adhesin that is under the
transcriptional control of Bcr1, partly but significantly rescued the bcr1/bcr1 biofilm phenotype in vivo. Since HWP1
overexpression only partly reversed the biofilm phenotype, we investigated whether additional mechanisms, besides
adhesin down-regulation, were responsible for the reduced virulence of this mutant. We discovered that the bcr1/bcr1
mutant was more susceptible to damage by human leukocytes when grown on plastic or on the surface of a human oral
mucosa tissue analogue. Overexpression of HYR1, but not HWP1, significantly rescued this phenotype. Furthermore a hyr1/
hyr1 mutant had significantly attenuated virulence in the mouse oral biofilm model of infection. These discoveries show that
Bcr1 is critical for mucosal biofilm infection via regulation of epithelial cell adhesin and neutrophil function.
Citation: Dwivedi P, Thompson A, Xie Z, Kashleva H, Ganguly S, et al. (2011) Role of Bcr1-Activated Genes Hwp1 and Hyr1 in Candida Albicans Oral Mucosal
Biofilms and Neutrophil Evasion. PLoS ONE 6(1): e16218. doi:10.1371/journal.pone.0016218
Editor: Dana Davis, University of Minnesota, United States
Received August 24, 2010; Accepted December 10, 2010; Published January 25, 2011
Copyright: ? 2011 Dwivedi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These studies were supported by National Institutes of Health grants RO1DE013986, RO1DE013986-08S1 (ADB) and R01AI067703 (APM). The funders
had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Oral pseudomembranous candidiasis (thrush) is the most
prevalent form of Candida infection in patients with weakened
or immature immune systems, such as HIV+ children, neonates
and patients with malignancies [1,2,3]. A resurgence of oral thrush
in children was recently reported due to the rising use of inhaled
corticosteroids, affecting up to 40% of children after long term
treatment . Surprisingly, up to 15% of children with no
underlying immune abnormalities present with oral thrush lesions
in the pediatric practice .
Pseudomembranous candidiasis is one of several clinical forms
of Candida infection and has distinct clinical and histopathological
characteristics. Clinically this infection presents as white plaques
on the oral mucosa, which can be removed by gentle rubbing .
These pseudomembranes were recently recognized as archetypal,
complex tissue biofilms and were proposed to be responsible for
the recalcitrant nature of this infection [7,8]. Using a mouse model
of oral thrush we characterized these biofilms and discovered that
they are complex, comprising of yeast, hyphae, commensal
bacteria, and neutrophils that form nests within the biofilm mass
. Both host and fungal-derived products fill the intercellular
spaces, thus forming a supporting biofilm matrix . Although
several C. albicans gene products have been implicated in biofilm
development on abiotic surfaces [10,11,12,13,14,15], information
on genes that enable biofilm formation on mucous membranes has
only recently begun to emerge .
The transcription factor Bcr1 governs biofilm formation in vivo
in the catheter, denture and vaginal models [16,17,18]. Although
Bcr1 is not required for hyphal morphogenesis, it acts as a positive
regulator of hyphal-specific adhesins [11,18]. Manipulation of
Bcr1 downstream target genes through mutation and overexpres-
sion showed that the surface adhesins Als3 and Hwp1 significantly
contribute to biofilm formation in the catheter model. Because
biofilm formation on abiotic and biological surfaces may be
regulated by similar processes we hypothesized that a bcr1/bcr1
mutant may also be defective in oral mucosal biofilm develop-
ment. Using both in vivo and in vitro models we tested the ability
of this mutant to form biofilms on the oral mucosa and dissected
the specific contribution of Bcr1-regulated genes in this phenotype.
Results and Discussion
To study the contribution of Bcr1-regulated genes in mucosal
biofilms, a mouse oral infection model was used where C. albicans
forms white pseudomembranes (biofilms) on the dorsal surface of
the tongue . Tongues from animals infected with genetically
manipulated strains were excised and examined by macroscopic
‘‘clinical’’ evaluation, assessment of cultivable fungal burden, and
histologic analysis to visualize the thickness of biofilms. Consistent
PLoS ONE | www.plosone.org 1January 2011 | Volume 6 | Issue 1 | e16218
with results in the mouse vaginal mucosa model , we found
that the bcr1/bcr1 strain was deficient in forming a clinically visible
mucosal biofilm on the tongues of immunocompromised mice in
vivo (Fig. 1). At the histologic level this mutant formed a thin,
interrupted biofilm on the dorsal surface of the tongue (Fig. 1,
arrows). These results are in agreement with the recently reported
attenuated biofilm phenotype of a bcr1/bcr1 mutant in the rat
denture biofilm model .
Surface area estimates of pseudomembranes, examined macro-
scopically during necropsy, showed approximately 80–100%
coverage of the tongue dorsal surface with biofilm formed by the
reference and reconstituted strains, while less than 10% of the
tongue surface in mice infected with the bcr1/bcr1 mutant was
covered by biofilm (Fig. 2A). In accordance with this we also found
that the tongue fungal burden of mice infected with the bcr1/bcr1
mutant was significantly lower than that of mice infected with
either the reference or complemented strains (Fig. 2B).
C. albicans also forms a biofilm when grown on a three-
dimensional model of the human oral mucosa . When grown on
this model, the bcr1/bcr1 mutant was slow in forming a mature
biofilm, and after 24 hours of growth formed a biofilm comprised
mainly of yeast cells, in contrast to its reference and complemented
strains (Fig. 3). Moreover, this mutant was significantly less capable
of triggering mucosal tissue damage (Fig. 4). This could be
attributed to the dominant yeast morphotype in biofilm cells,
which lacks expression of several hyphae-specific epithelial
adhesins that may also be involved in oral epithelial cell damage
Adhesion is a fundamental process under Bcr1 control that
promotes biofilm formation on catheter surfaces . Therefore,
we evaluated the contribution of the Bcr1-regulated adhesins Als1,
Als3 and Hwp1 in the capacity of C. albicans to form a biofilm on
the oral mucosa in vivo. Increased expression of ALS1 in the bcr1/
bcr1 mutant background did not significantly affect the surface
area covered by biofilm (Fig. 2A), increase the tongue fungal
Figure 1. Biofilm formation and histological examination of the tongues of mice infected with the bcr1/bcr1 mutant, DAY185
(reference) and complemented strains. Tongues of immunocomrpomised animals were excised after five days of infection and the dorsal aspect
was digitally photographed. Four mice were infected with each strain and representative clinical pictures are shown from 1 mouse in each group on
the left panel. On the right panel, representative PAS-stained thin sections of the tongue of one mouse per group are shown. Arrows indicate the
Figure 2. Biofilm surface area and fungal burden of animals
infected with the bcr1/bcr1 mutant and related strains. (A)
Percent tongue surface area covered by biofilm. Results represent the
average of 4 tongues in each group. Image J was used to calculate the
area covered by white plaques as well as the total surface area of each
tongue. Error bars represent standard deviations. *p=0.0000 for bcr1/
bcr1 mutant versus reconstituted strain, **p=0.026 for bcr1/bcr1TEF-
HWP1 versus bcr1/bcr1 mutant strain. (B) Tongue fungal burden. Results
represent the average of four mice per group and error bars represent
standard deviations. *p=0.0004 for bcr1/bcr1 mutant versus reconsti-
tuted strain, **p=0.0002 for bcr1/bcr1TEF-HWP1 versus bcr1/bcr1
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burden (Fig. 2B), or promote mucosal biofilm formation at the
histologic or macroscopic level (Fig. 5). Similarly, expression of
ALS3 under the TEF1 promoter did not significantly affect the
surface area covered by biofilm (Fig. 2A), the tongue fungal
burden (Fig. 2B), or the biofilm thickness (Fig. 5, arrows); the
strain’s intermediate phenotype is discussed further below. A clear
example of phenotypic rescue from overexpression was observed
with HWP1. Specifically, HWP1 overexpression in this genetic
background improved biofilm formation and increased fungal
burden in the animals significantly (Fig. 2A, 2B, 5). This finding
agrees with observations in the catheter biofilm model where this
construct partly but significantly reversed the bcr1/bcr1 phenotype
. In addition to being important in biofilm development
[12,22], Hwp1 is also a well established oral epithelial cell adhesin
, which plays a role in the pathogenesis of oral infection
[23,24]. Our findings provide additional support for the role of
Hwp1 in oral infection, and in addition argue that the reduced
expression of HWP1 in the bcr1/bcr1 mutant is a major cause of the
mutant’s oral epithelial biofilm defect.
ALS3 overexpression in the bcr1/bcr1 background partly
reversed the bcr1/bcr1 phenotype, based upon visual inspection
(Fig. 5). However, the mean percentage surface area covered by
biofilm and the CFU counts from infected tissue with this strain
did not reach statistical significance (Fig. 2A, 2B). These findings
were thus somewhat in contrast with data in the venous catheter
biofilm model showing that, although Als3 is not absolutely
required for biofilm formation, ALS3 overexpression completely
reverses the biofilm phenotype of the bcr1/bcr1 mutant . We
note that these two biofilm experimental systems are quite
different, and that C. albicans adhesins exhibit high substrate
specificity, even when they have highly related sequences in their
binding domains . In addition, innate host defense mecha-
nisms in the oral cavity, coupled with salivary flow and mechanical
cleansing by chewing, may modify the ability of these strains to
establish a biofilm in vivo.
However, because regulation of biofilm-associated gene expres-
sion may vary significantly in different biofilm model systems ,
we wanted to rule out the possibility that Als3 expression is Bcr1-
independent in the oral mucosa, which could explain the disparate
results with these strains in different model systems. Therefore, we
quantified Als3 gene expression in the bcr1/bcr1 mutant and
adhesin-overexpressing strains, when grown on a three dimen-
sional model of the human oral mucosa. As anticipated, when
grown on an oral mucosa tissue analogue Als3 expression levels
were lower in the bcr1/bcr1 deletion mutant as well as in the ALS1-
and HWP1-overexpressing strains, compared to the reference
strain (Fig. 6). In contrast, Als3 expression levels in the ALS3-
overexpressing strain were three fold higher than the reference
strain (Fig. 6). This finding argues against the possibility of
differential regulation of Als adhesins in the oral mucosa, and
further supports the idea that different experimental systems can
reveal tissue-specific functions of adhesins. Thus our results
Figure 3. Biofilm formed by the bcr1/bcr1 mutant, complemented and reference (DAY185) strains on a three-dimensional
organotypic model of the oral mucosa. Histologic pictures show 406magnification after 6 and 24 hours of inoculation. Thin sections were
stained with PAS. Arrows indicate biofilms forming on the (apical) epithelial surface of the cultures. Results are representative of one of three
Figure 4. Mucosal damage by the indicated strains in the three
dimensional model of the oral mucosa. Cell damage by the bcr1/
bcr1 mutant and reference strains was quantified by the release of
lactate dehydrogenase (LDH) in the media. Results are the mean 6 SD
of three experiments, each condition set up in triplicate. *bcr1/bcr1
mutant significantly different from DAY185 (reference) strain, p=0.001–
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establish that Hwp1, and not Als3, is a critical Brc1 adhesin target
relevant to oral thrush. In agreement with our findings, it has been
reported that biofilm formation in a subcutaneous rat model
requires Bcr1 but not Als3 .
Due to the complexity of the structure of oral mucosal biofilms
, transcriptional deregulation of a single C. albicans gene is
unlikely to be responsible for mediating loss of the mucosal biofilm
phenotype in the bcr1/bcr1 mutant. This explains the finding that
HWP1 overexpression only partially reversed the bcr1/bcr1
phenotype. We thus sought to identify additional, adhesion-
unrelated genes under the transcriptional control of Bcr1,
contributing to its inability to form mucosal biofilms.
A prominent feature of infections characterized by soft tissue
biofilms is infiltration of infected tissues by neutrophils, which
confer innate immune protection [27,28]. We previously showed
that neutrophils infiltrate the oral mucosal biofilm mass in this
mouse oral infection model . We thus hypothesized that the
bcr1/bcr1 mutant fails to develop a robust biofilm on the oral
mucosa at least partly because it is more efficiently cleared by
biofilm-infiltrating neutrophils. To begin to test this hypothesis we
examined susceptibility of this mutant to killing on plastic by the
HL-60 neutrophil-like cell line using a modification of the XTT
assay . Indeed, we found that the bcr1/bcr1 mutant was more
susceptible to killing than its reference and reconstituted strains,
regardless of the effector to target ratio used in killing assays
(Fig. 7). In fact the effector to target ratio of the bcr1/bcr1 strain
corresponding to the MIC50 in this assay was five times lower than
the reference and reconstituted strains (Fig. 7). These findings were
confirmed when freshly isolated human neutrophils were used in
killing assays (Fig. 8A–B). Finally, we extended these findings to
the oral mucosa, by testing susceptibility of the bcr1/bcr1 mutant to
leukocyte killing on a three-dimensional model of the human oral
mucosa (Fig. 8C). As expected, this mutant was also more
susceptible to leukocyte-inflicted damage when grown on a three
dimensional model of the human oral mucosa (Fig. 8C),
supporting our hypothesis that it may be more effectively cleared
in the oral environment.
Overexpression of HYR1, but not HWP1, in the bcr1/bcr1
background, significantly rescued the higher susceptibility pheno-
type of this mutant to killing by leukocytes both on plastic and on
the oral tissue surface (Fig. 8A–C). These findings confirmed
Figure 5. Biofilm formation and histological examination of the tongues of mice infected with strains overexpressing adhesins
ALS1, ALS3 and HWP1 in the bcr1/bcr1 background. Tongues of immunocomrpomised animals were excised after five days of infection and the
dorsal aspect was digitally photographed. Four mice were infected with each strain and representative clinical pictures are shown from 1 mouse in
each group on the left panel. On the right panel, representative PAS-stained thin sections of the tongue of one mouse per group are shown. Arrows
indicate the biofilm thickness.
Figure 6. Detection of Als3 expression in the bcr1/bcr1 mutant,
reference (DAY185) and adhesin-overexpressing strains on the
3D model of the human oral mucosa. The indicated strains of C.
albicans were grown for 24 h and C. albicans RNA was extracted. The
relative transcript levels of Als3 were measured by real-time PCR.
Results are the mean 6SD of three biological replicates, each tested in
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previous reports showing that Hyr1, but not Hwp1 is involved in
susceptibility to neutrophil killing [30,31]. More importantly, these
data, combined with the finding that the hyr1/hyr1 mutant was
highly susceptible to killing in an oral mucosal tissue model
(Fig. 8C), suggest that this gene is indirectly contributing to the
observed mucosal biofilm phenotype by conferring resistance to
neutrophil killing. Thus, this study now strongly implicates Hyr1 in
innate immune cell evasion of C. albicans in the oral mucosa.
Hyr1 is a GPI-anchored cell wall protein, expressed during
hyphal development  and repressed upon neutrophil encoun-
ter [30,33], however, relatively little is known about its exact role
in virulence. Because there is no severe biofilm defect on catheter
surfaces in the hyr1/hyr1 mutant , we hypothesized that this
mutant may have only a moderately attenuated biofilm phenotype
on the tongue surface. As expected, this mutant formed biofilms
covering part of the tongue surface, and overexpression of HYR1
in the bcr1/bcr1 background did not rescue the oral mucosal
biofilm phenotype (Fig. 9, 10A). However, we also anticipated that
due to increased susceptibility to neutrophil killing, preventing
deep tissue invasion , the tissue fungal burden in mice infected
with the hyr1/hyr1 mutant would be severely attenuated.
Consistent with this hypothesis, histological assessment showed
that, in contrast to the reference strain which reached the granular
Figure 7. Susceptibility of the bcr1/bcr1 mutant, reference
(DAY185) and reconstituted strains to leukocyte-inflicted
damage. Strains were exposed to HL-60 cells that had been
differentiated into neutrophil-like cells in vitro. HL-60 cells were added
to C. albicans for 3 h at effector to target cell ratios (E:T) ranging from
5:1 to 1:2. Results represent the mean 6 SD of three experiments, each
condition set up in triplicate.
Figure 8. Susceptibility of the bcr1/bcr1 mutant, hyr1/hyr1 mutant, and HYR1- or HWP1- overexpressing strains in the bcr1/bcr1
background, to human leukocytes. Susceptibility was tested on 96 well plates (A,B) or on a three dimensional model of the human oral mucosa
(C). Strains were exposed to differentiated HL-60 cells (A,C) or freshly isolated neutrophils from one human donor (B) at an effector to target cell ratio
of 1:1 (A,B) or 10:1 (C). Results represent the mean 6 SD of three experiments, each condition set up in triplicate. *p,0.03 and **p,0.05 for a
comparison between the bcr1/bcr1 mutant and HYR1- overexpressing strain in the bcr1/bcr1 background.
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and prickle epithelial cell layers, the hyr/hyr1 mutant was confined
within the keratin layer (Fig. 9 arrows). As a result, the tissue fungal
burden was severely attenuated in mice infected with the hyr1/hyr1
mutant (Fig. 10B). However, overexpression of HYR1 in the bcr1/
bcr1 background was not sufficient to reverse the tissue invasion or
fungal burden phenotype of the bcr1/bcr1 mutant (Fig. 9 arrows,
and 10B). This finding is consistent with the fact that, in addition
to neutrophil clearance, other virulence mechanisms directly
related to epithelial adhesion and invasion, as noted above, are
contributing to the severely attenuated phenotype of this mutant.
In conclusion, our studies provide valuable new insights which
promote the understanding of the pathogenesis of Candida
mucosal biofilm infections. We have shown that the transcription
factor Bcr1 is a critical regulator of oral mucosal biofilm formation
and identified two genes, HWP1 and HYR1, under Bcr1 control
that govern the inability of the bcr1/bcr1 strain to form robust oral
mucosal biofilms. Although much is known about the function of
Hwp1 and its role in oral mucosal infection, this is the first study
implicating Hyr1 in the pathogenesis of oral biofilm infection.
The study was approved by the University of Connecticut
Health Center Animal Care Committee (Protocol Number: 2009-
541) and the Human Subjects Protection Office (IRB Number:
02-288-2). Animals were monitored daily for distress. Given that
the oral cavity is readily accessible, lesions are detected relatively
early in their onset and animals are euthanized after lesion
formation before visible distress/behavior signs are observed. A
written informed consent was signed by all healthy human blood
C. albicans strains
The C. albicans deletion mutants and overexpressing strains
and their construction are described in detail elsewhere [11,18].
Strain DAY185 is a His+reference strain used to construct the
His+bcr1/bcr1 deletion mutant used in all experiments [18,35].
Strain DAY286  was used as a reference strain for the hyr1/
hyr1 null mutant in some experiments. The two reference strains
were phenotypically similar in all experimental systems described
in this study. All strains exhibited similar growth characteristics
when grown overnight in epithelial cell media (not shown).
Mouse model of mucosal biofilm
Different strains were tested for their ability to form an oral
biofilm using our previously described mouse model . Briefly,
6–8 week old female C57BL/6 mice were immunosuppressed by
subcutaneous injection with cortisone acetate (225 mg/kg, dis-
solved in 200 ml PBS containing 0.5% Tween-20) on days -1, 1
and 3 relative to infection. To deliver the C. albicans challenge
mice were anaesthetized by an intramuscular injection of ketamine
and xylazine (90–100 mg/kg and 10 mg/kg of body weight,
respectively) and a small cotton pad soaked with 100 ml of C.
albicans cell suspension (66108cells/ml) was used to swab the
entire oral cavity. The swab was left for 2 h under the tongue and
was removed before the animals awoke. This procedure was
repeated 2 days later and mice were sacrificed after 5 days of total
exposure to C. albicans. During the infection period animals were
also given drinking water containing a daily-fresh suspension of
each strain (66106yeast organisms/ml) to maintain high oral
carriage loads throughout the experimental period. Tongues were
removed aseptically at necropsy, photographed, and images were
saved as jpg files. Images were subsequently analyzed using the
NIH Image J software (http://rsb.info.nih.gov/ij) and data were
Figure 9. Biofilm formation and histological examination of the tongues of mice infected with DAY286 (reference), hyr1/hyr1
mutant and HYR-1 overexpressing strains in the bcr1/bcr1 background. Tongues of immunocomrpomised animals were excised after five
days of infection and the dorsal aspect was digitally photographed. Four mice were infected with each strain and representative clinical pictures are
shown from 1 mouse in each group on the left panel. On the right panel, representative PAS-stained thin sections of the tongue of one mouse per
group are shown. Arrows indicate microorganisms invading the spinous cell layer of the epithelium (strain DAY286) or remaining superficially within
biofilms (hyr1/hyr1 mutant and HYR1- overexpressing strains).
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expressed as percent surface area covered by biofilm (total surface
area of white lesions/entire tongue dorsal surface area).
To determine the number of viable organisms in oral tissues, the
tongue was longitudinally dissected in three equal pieces and the
central portion was processed for colony forming unit (CFU)
quantification at the time of sacrifice. The tissue was weighed,
rinsed with phosphate buffered saline and then homogenized using
a tissue homogenizer. Serial dilutions were plated onto Sabouraud
dextrose agar plates containing 10 mg/ml chloramphenicol
(Sigma, USA) and plates were incubated at 30uC for 48 h. Results
were expressed as log CFU counts/g of tissue. One portion of the
longitudinally dissected tongues was fixed with 10% buffered
formalin and embedded in paraffin. Five mm thick sections were
prepared and stained with hematoxylin and eosin (H&E) and
Periodic Acid Schiff (PAS) stains.
Three-dimensional model of the oral mucosa
To investigate in vitro mucosal biofilm formation, tissue damage
by C. albicans and leukocyte-mediated fungal killing in an oral-like
environment we used a three-dimensional model of the oral
mucosa as previously described . This system is composed of
3T3 fibroblasts embedded in a biomatrix of collagen type I,
overlaid by a multilayer of well-differentiated oral epithelial cells
(OKF6/TERT-1). C. albicans cells (16106yeast cells) were added
to the cultures apically in 100 ml of airlift medium without FBS
and antibiotics. In some experiments, after 6–44 hours of co-
culture mucosal tissues were formalin-fixed, embedded in paraffin
and sections were stained with PAS. The extent of mucosal tissue
damage was quantified at different time points by measuring
extracellular leakage of LDH in the medium, using the cytotox-96
assay (Promega) as previously described . Leukocyte-mediated
fungal cell damage was assessed in this model as described below.
Assessment of susceptibility to neutrophil killing
The susceptibility of the different strains to a neutrophil-like cell
line (HL-60 cells) or to human freshly isolated peripheral blood
neutrophils was determined by the XTT assay, as previously
described . Briefly, HL-60 cells were cultured in RPMI 1640
medium containing 10% fetal bovine serum and 25 mM HEPES
and were induced to differentiate into neutrophil-like cells by
exposure to 1.25% of dimethyl sulfoxide for 7–9 days. Neutrophils
were isolated from anticoagulated blood of one healthy donor by
dextran T-500 (Sigma-Aldrich, St. Louis, MO) sedimentation
followed by Histopaque-1077 (Sigma-Aldrich) density gradient
centrifugation. Granulocyte-erythrocyte pellet was collected, and
erythrocytes were lysed by hypotonic shock. Neutrophils were
washed with HBSS without Ca, Mg (Mediatech, Inc., Herndon,
VA) and resuspended in RPMI1640 (Mediatech, Inc.) with
Figure 10. Biofilm surface area and fungal burden in animals infected with the bcr1/bcr1 mutant, hyr1/hyr1 mutant, HYR1-
overexpressing and reference strains. (A) Percent tongue surface area covered by biofilm. Results represent the average of 4 tongues in each
group. Image J was used to calculate the area covered by white plaques as well as the total surface area of each tongue. Error bars represent standard
deviations. *p=0.015 for hyr1/hyr1 mutant versus reference strain; **p=0.008 for hyr1/hyr1 mutant versus bcr1/bcr1TEF-HYR1 strain; ***p=006 for
bcr1/bcr1TEF-HYR1 versus reference strain. (B) Tongue fungal burden. Results represent the average of four mice per group and error bars represent
standard deviations. *p=0.000 for bcr1/bcr1TEF-HYR1 or hyr1/hyr1 compared to reference strain, **p=0.23 for hyr1/hyr1 versus the bcr1/bcr1TEF-HYR1
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10 mM HEPES (Gibco Invitrogen, Grand Island, NY). The
resulting cell preparations consisted of more than 95% of
neutrophils by Wright-Giemsa stain and were more than 98%
viable by trypan blue exclusion.
To perform Candida killing assays on plastic, overnight YPD
broth cultures of C. albicans were resuspended in DMEM 10%
fetal bovine serum and were added to 96 well plates (100 ml/well)
at concentrations ranging from 105to 26104cells/well. There was
a linear relationship between viable cell number and colorimetric
signal (XTT activity) in this concentration range with all strains
(not shown). Immune cells were added to C. albicans at effector to
target cell ratios (E:T) ranging from 5:1 to 1:2. To perform
Candida killing assays in an oral-like environment C. albicans cells
were grown as above and added apically to the three-dimensional
model of the oral mucosa (16105yeast cells/tissue), in 50 ml of
airlift medium without FBS and antibiotics. After a 4 h incubation
period, differentiated HL-60 cells were added apically at an
effector to target ratio of 10:1 and incubated for 3 more hours.
After incubation of Candida with effectors at 37uC, 5% CO2for
3 hours, media were aspirated and mammalian cells were lysed
with sterile H2O. 100 ml/well of XTT solution containing
Coenzyme Q0 (0.25 mg/ml XTT and 40 mg/ml coenzymeQ0)
was added to each well and plates were incubated at 37uC and
5%CO2for 2 hrs. Supernatants were transferred into new plates,
and optical densities (OD) were measured by an Opsys Microplate
Reader (Thermo Labsystems, Franklin, MA) at 450–490 nm, with
a 630 nm reference filter. Antifungal activity was calculated
according to the following formula: %fungal damage=(12x/
n)*100, where x is the OD450 of experimental wells (C. albicans
with effectors) and n is the OD450 of control wells (C. albicans
To test expression of the Bcr1-regulated gene ALS3 in an oral
model of infection, we quantified expression by real-time RT-PCR
after 24 h growth on the 3D model of the human oral mucosa.
Briefly, media were aspirated and the collagen gel was transferred
to a centrifuge tube, followed by a brief centrifugation at 4uC.
Next, the transferred gel was dissolved in mammalian cell RNA
extraction buffer (4 M guanidine thiocyanate, 25 mM sodium
citrate, 0.5% Sarkosyl [N-lauroyl-sarcosine], and 0.1 M Beta-
mercaptoethanol), and repeatedly passed through a 20K gauge
needle . The organisms were spun in a centrifuge at 14 0006g
at 4uC, and then snap-frozen in liquid nitrogen. Fungal RNA was
isolated using the RiboPure yeast kit (Ambion, Inc.), according to
the manufacturer’s instructions. RNA was reverse transcribed with
oligo(dT) primers using Superscript reverse transcriptase II
Primers used for measurement of transcript levels were as
follows. We used the sequences described by Green et al.  for
measuring ALS3 RNA levels: ALS3 FOR: 59-CCACTTCA-
CAATCCCCATC-39, and ALS3 REV: 59-CAGCAGTAGTAG-
TAACAGTAGTAGTTTCATC-39. We used sequences de-
scribed by Blankenship et al.  for measuring control TDH3
RNA levels: TDH3 FOR: 59-AAATCGGTGGAGACAACAGC-
39, and TDH3 REV: 59-TGCTAAAGCCGTTGGTAAGG-39.
RT-PCR reaction conditions were as follows: 26 iQ SYBR
Green Supermix (Bio-Rad), 1 ml of first-strand cDNA reaction
mixture, and 0.1 mM of primers were mixed in a total volume of
50 ml per reaction. Real-time PCR was carried out in duplicate for
each sample using the iCycler iQ real-time PCR detection system
(Bio-Rad). The program for amplification included an initial
denaturation step at 95uC for 5 min, followed by 40 cycles of 95uC
for 45 s and 58uC for 30 s. Product amplification was detected
using SYBR Green fluorescence during the 58uC step. The
reaction specificity was monitored by melt-curve analysis. TDH3
was used as a reference gene for normalization of gene expression,
which was done using Bio-Rad iQ5 software (DDCTmethod).
Conceived and designed the experiments: ADB APM. Performed the
experiments: PD AT HK ZX SG. Analyzed the data: PD AT HK SG.
Contributed reagents/materials/analysis tools: APM. Wrote the paper:
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