This document describes the development of a new high-throughput screening method to identify inhibitors of the yeast-to-hypha morphological transition in the fungal pathogen Candida albicans. The method uses automated microscopy and image analysis to quantitatively measure morphological parameters that distinguish between yeast and hyphal forms. Parameters like length/width ratio and mean object shape are calculated from images of fluorescently stained cells. Cell viability is also measured to identify inhibitors that block morphological transition without affecting growth. The method is validated using known inhibitors like farnesol and shown to be suitable for high-throughput screening through calculation of the Z-factor metric. This screening method could help identify new antifungal drug candidates that target virulence traits rather

1. Journal of Biomolecular Screening
2015, Vol. 20(2) 285­–291
© 2014 Society for Laboratory
Automation and Screening
DOI: 10.1177/1087057114552954
jbx.sagepub.com
Technical Note
Introduction
Candida albicans is the most common human fungal patho-
gen, even though it is part of the commensal microflora in the
gastrointestinal and urogenital tracts as well as in the oral
cavity. Vaginal candidiasis has been described at least once in
75% of all women worldwide.1
Although the disease is not
invasive, the quality of life of affected individuals is consid-
erably reduced. Notably, up to 10% of these women addition-
ally experience relapsing incidents of vulvovaginal
candidiasis. Bloodstream infections with Candida spp. (can-
didemia) have been increasing in hospital intensive care units
(ICUs) worldwide, with an annual prevalence of 300,000
cases and 30% to 50% mortality.2,3
Among systemic micro-
bial infections in ICU patients, candidiasis ranks second in
Europe and North America, causing more than 50% of the
cases.2
Despite the increase in Candida infections, currently
available therapeutic agents remain few in number, and of
those, several can lead to severe side effects, such as liver
damage.4
The most important antifungal drugs can be classified into
six categories with confined fungal targets.5
Polyenes bind
ergosterol and induce pores in fungal plasma membranes.
Azoles and allylamines inhibit the synthesis of ergosterol,
rendering the membranes unstable, whereas morpholines and
antimetabolites prevent nucleic acid production. Many cur-
rently applied antifungal drugs have severe side effects.4
The
ergosterol-attacking agents, for instance, can additionally
interfere with the human analog cholesterol, resulting in host
cell damage. Thus, long-term use of these drugs can cause
renal dysfunction, liver toxicity, or bone marrow depression.
552954JBXXXX10.1177/1087057114552954Journal of Biomolecular ScreeningStylianou et al.
research-article2014
1
Department of Clinical Microbiology, Umeå University, Umeå, Sweden
2
Umeå Centre for Microbial Research (UCMR), Umeå, Sweden
3
Laboratory for Infection Medicine Sweden (MIMS), Umeå University,
Umeå, Sweden
4
Department of Chemistry, Umeå University, Umeå, Sweden
Received Jun 30, 2014, and in revised form Sep 2, 2014. Accepted for
publication Sep 4, 2014.
Corresponding Author:
Constantin F. Urban, Department of Clinical Microbiology, Umeå
University, 90185 Umeå, Sweden.
Email: constantin.urban@umu.se
Novel High-Throughput Screening
Method for Identification of Fungal
Dimorphism Blockers
Marios Stylianou1,2,3
, Hanna Uvell2,4
, José Pedro Lopes1,2,3
,
Per-Anders Enquist2,4
, Mikael Elofsson2,4
, and Constantin F. Urban1,2,3
Abstract
Invasive mycoses have been increasing worldwide, with Candida spp. being the most prevalent fungal pathogen causing high
morbidity and mortality in immunocompromised individuals. Only few antimycotics exist, often with severe side effects.
Therefore, new antifungal drugs are urgently needed. Because the identification of antifungal compounds depends on
fast and reliable assays, we present a new approach based on high-throughput image analysis to define cell morphology.
Candida albicans and other fungi of the Candida clade switch between different growth morphologies, from budding yeast to
filamentous hyphae. Yeasts are considered proliferative, whereas hyphae are required for invasion and dissemination. Thus,
morphotype switching in many Candida spp. is connected to virulence and pathogenesis. It is, consequently, reasonable to
presume that morphotype blockers interfere with the virulence, thereby preventing hazardous colonization. Our method
efficiently differentiates yeast from hyphal cells using a combination of automated microscopy and image analysis. We
selected the parameters length/width ratio and mean object shape to quantitatively discriminate yeasts and hyphae. Notably,
Z′ factor calculations for these parameters confirmed the suitability of our method for high-throughput screening. As a
second stage, we determined cell viability to discriminate morphotype-switching inhibitors from those that are fungicidal.
Thus, our method serves as a basis for the identification of candidates for next-generation antimycotics.
Keywords
mycoses, high-throughput screening, Candida, yeast-to-hypha transition, antifungal compounds
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2. 286 Journal of Biomolecular Screening 20(2)
Finally, echinochadins constitute a new group of antifungal
agents. They are inhibitors of glucan synthetase in the cell
wall of fungi, which has no counterpart in human hosts,
resulting in less severe side effects. However, emergence of
echinocandin-resistant strains is a cause of clinical concern.
Systemic and superficial candidiasis is strictly associ-
ated with the reversible morphotype switching from bud-
ding yeasts to the filamentous hyphae (Y-H).6
The yeast is a
unicellular morphotype and considered the commensal
form of C. albicans.7
Derogation of innate or adaptive
immunity can induce adherence to epithelia and conversion
from yeast to hyphal growth.6
Hyphal growth results in a
filamentous morphology initiated by germ tube formation
at a yeast mother cell. Apical growth at the tip of the fila-
ment continues with occasional branching events. Hyphae
have been reported to be essential for invasion and dissemi-
nation to noncommensal niches as well as for biofilm for-
mation and escape from host immune cells.6
Notably,
biofilms are a major cause of medical device failure and a
frequent source of relapsing infections.8
C. albicans tran-
scription factor knockout mutants (for instance, Δedt1 and
Δefg1) that are locked in the yeast morphology have been
shown to be nonvirulent in animal models and to be unable
to form biofilms, supporting the concept of morphotype
transition as a virulence trait.8–10
We reasoned that identifi-
cation of compounds that inhibit the Y-H transition can be
exploited for application as antifungal therapy. Fungal
growth should not be altered by these agents but rather redi-
rected into a commensal state that can be controlled by the
immune system. According to these assumptions, we
hypothesize that during treatment with such agents, selec-
tion pressure on fungal pathogens is low and, thus, in turn
the possibility for resistance development decreased.
Therefore, we aimed to develop a method to screen large
chemical compound libraries for Y-H transition inhibitors.
The ideal compounds are those that prevent C. albicans
morphotype transition without affecting the cellular viabil-
ity of fungal cells.
The method is based on automated microscopic imaging
of labeled fungal cells and thereafter quantitative image
analysis, referred to as high-content analysis (HCA). Using
HCA, we calculated the mean object shape (MOS) and
length/width ratio (LWR) of fungal cells. MOS and LWR
were selected to define and quantify the Y-H transition, as
calculated by eq 1 (see the Materials and Methods section).
To identify compounds that are fungistatic or fungicidal, we
quantified fungal viability by measuring adenosine triphos-
phate (ATP) levels, an assay we previously applied to screen
drug libraries for antifungal activity.5
Viability was calcu-
lated using eq 2 (see the Materials and Methods section).
To verify our method, we used farnesol, a natural quorum-
sensing molecule secreted by C. albicans. Farnesol blocks
hyphal growth without affecting the proliferation as yeast-
form cells.8
In addition, we used knockout-mutant strains
that are restricted to yeast-form growth, namely, Δedt110
and Δefg1.9
These transcription factor knockout strains are
unable to switch from yeast form to hyphal growth, even
when growing in otherwise hypha-inducing conditions.
Thimerosal served as a reference for fungistatic or fungi-
cidal agents. The mercury compound kills fungal cells by
disruption of mitochondria. Furthermore, the Z′ factors for
quality assessment of the method were calculated for both
LWR and MOS at 6 h using eq 3 (see the Materials and
Methods section), defining our method as valid and suitable
for high-throughput screening.11
Materials and Methods
Media and Cultivation of Fungi
Conditions and cell concentrations were based on the
antifungal susceptibility testing (AFST) guidelines of the
European Committee on Antimicrobial Susceptibility
Testing (EUCAST).12
C. albicans (SC5314) and C. albicans
engineered gene knockout mutant strains locked to yeast-
form growth, Δedt110
and Δefg19
(derived from parental
strain C. albicans CAI4), were used in this study.
Yeast cells were grown overnight by shaking at 30 °C in
synthetic complete dropout medium with 2% glucose (SC).
Subcultures were inoculated from an overnight culture to a
final concentration of 107
cells/mL in SC and incubated for
4 h at 30 °C. Cells were washed twice in phosphate-buff-
ered saline 1× (PBS) and adjusted to 2.0 × 105
cells/mL in
PBS. To each well of a black, 96-well plate with transparent
bottom (Costar, Fisher Scientific, Waltham, MA), 150 µL
RPMI 1640 with 0.5% DMSO (0.5%) followed by 50 µL
yeast suspension were added using a WellMate Matrix
(Thermo Scientific, Waltham, MA) to give a final volume
of 200 µL in each well. The plate was then incubated at 37 °C
at 5% CO2
for 3, 6, or 24 h. Under these conditions, C. albi-
cans yeast cells start to germinate and to grow as hyphae. To
keep the cells at the bottom of the well for better micro-
scopic imaging, the wells were pretreated with 0.1% w/v
poly-L-lysine (Sigma-Aldrich, St. Louis, MO) for 30 min
and washed three times with PBS. In addition to the above
conditions, we added 250 µM farnesol (Sigma-Aldrich) or
0.8% w/v thimerosal (Sigma-Aldrich) to SC5314 as refer-
ences for morphotype switching inhibition and fungicidal
activity, respectively.
Determination and Quantification of Y-H Switch
Using HCA
After incubation for 3, 6, and 24 h, C. albicans cells were
fixed with 2% paraformaldehyde and stained with 0.1% cal-
cofluor white (CFW; Sigma-Aldrich). We used the chitin-
specific fluorescent dye CFW to analyze the fluorescent
signal of stained fungal cell walls. Images were captured
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3. Stylianou et al. 287
with an automated microscope (HCA-Cellomics ArrayScan
VTI, Thermo Scientific) and the C. albicans cell morphol-
ogy analyzed. Based on HCA information, the individual
fungal cell morphotype was determined by means of LWR
and MOS, respectively. These two parameters were suffi-
cient to reliably discriminate between yeast and hyphal cul-
tures (eq 1). LWR determines the average ratio between
length and width, which indeed changes considerably dur-
ing apical growth of a filament versus division of ellipsoid
yeast cells by budding. MOS refers to the average measure
of detected objects based on the formula MOS =
[(c2
/4π)*area], which is the ratio of circumference squared
to 4π*area (MOS = 1 = circular object).
Determination of Cell Viability Using ATP
Quantification
We performed the cellular viability test prior to cell fixation
and chitin staining. The percentage of cellular viability was
determined using the CellTiter-Glo luminescent cell viabil-
ity assay (CTG; Promega, Madison, WI) to identify com-
pounds that are fungistatic or fungicidal. A volume of the
CTG reagent equal to the cell volume per well was added.
After 15 min at room temperature, the luminescence signal
was quantified in a luminometer (Infinite F200, Tecan,
Männedorf, Switzerland). The luminescence signal corre-
sponds to ATP values and thus to cellular viability. The per-
centage of cellular viability was calculated for the four
tested conditions, C. albicans either with farnesol (250 µM)
or thimerosal (0.8%) and the two mutant C. albicans strains
in comparison with C. albicans in DMSO (0.5%) as the
100% hyphal growth control (eq 2). The assay was per-
formed at least to three biological replicates in triplicate
(n = 3[3]) in 96-well plates with clear bottoms. Liquid han-
dling, plate reading, and automated microscopy were per-
formed at Laboratories for Chemical Biology Umeå
(LCBU), Chemical Biology Consortium Sweden (CBCS).
Calculations and Statistical Analysis
Growth inhibition (GrIn) was determined, after 3 and 6 h,
from the MOS and LWR values for all conditions. MOS and
LWR calculations are derived from the average number of
fluorescent pixels from at least 100 cells. The percentage
of GrIn (%GrIn
) was defined as eq 1: [%GrIn
=100 – ( x test
/
x DMSO
)*100]. Furthermore, the switching inhibition (SwIn)
(%SwIn
) was calculated from ATP values as eq 2: [%SwIn
=
100 – ( x test
/ x DMSO
)*100]. Thus, in high-throughput
screenings, the positive hits including growth and morphot-
ype inhibitors are determined using GrIn calculations. The
discrimination of growth from morphotype inhibitors is
defined by the SwIn formula. GrIn and SwIn calculations
were performed in Graphpad Prism 5.0 and analyzed for
statistical significance using a one-way analysis of variance
and Tukey’s multiple comparison test from at least three
biological replicates in triplicate (n = 3[3]) and applied for
0.5% DMSO (SC5314, Δedt1 and Δefg1), farnesol (250
µM), and thimerosal (0.8%). Moreover, the method validity
is defined by the Ζ′ factor as eq 3: [Ζ′ = 1 – [3*(SDDMSO
+
SDtest
)/(ABS( xDMSO
– x test
))]. The Ζ′ factors represent the
mean values from the calculation of at least three biological
replicates in triplicate (n = 3[3]).
Results and Discussion
A crucial virulence trait of polymorphic fungi is their ability
to reversibly switch from yeast-like to filamentous growth.
Hence, the aim of the study was to develop a reliable high-
throughput screening method for the identification of mol-
ecules that break the Y-H transition without disturbing cell
viability. Images gathered from an automated fluorescence
microscope were analyzed on the basis of fluorescent pix-
els. From the substantial amount of parameters created by
HCA, we chose LWR and MOS, because these values were
sufficient to reliably distinguish between yeast and hyphal
morphotypes (Fig. 1). This means in particular that C. albi-
cans samples with LWR and MOS values less than 1.5 are
defined as yeast cells (Fig. 1). After a 24 h incubation, LWR
and MOS values from the hyphal reference samples cannot
be taken into account, as confluent growth renders analysis
unfeasible. Microscopic images are nevertheless available
in substantial amounts for cell morphotype evaluation (data
not shown).
To validate whether our method is suitable for identify-
ing switching inhibitors, we used the quorum-sensing mol-
ecule farnesol. This natural compound prevents hyphal
growth of C. albicans under otherwise hyphae-inducing
conditions (Figs. 1–3). After 24 h of incubation with farne-
sol, however, C. albicans yeast growth was additionally
reduced to low levels, indicating that over long incubation
times, farnesol has growth-inhibitory activity. This is in
good agreement with a previous report that showed that
farnesol challenge of yeast cells prevented hyphal growth
but at the same time significantly reduced cellular viability.8
Farnesol is nevertheless a suitable reference for morphot-
ype switching inhibitors, because it does not affect C. albi-
cans growth within 6 h incubation periods (Fig. 3). We next
used thimerosal to kill off C. albicans cells, which after-
ward remain as dead and thus nonswitching yeasts. LWR
and MOS obtained from HCAdata confirmed that thimerosal-
treated C. albicans remained as yeasts, because values were
less than 1.5 and cellular viability was close to background
levels (Figs. 1–3). Thus, thimerosal could be used as a ref-
erence for fungicidal or fungistatic compounds.
We furthermore assayed two knockout C. albicans
mutants Δefg1 and Δedt1,9,10
both yeast-locked strains. They
serve as additional key references for determining the accu-
racy of discrimination between yeast growth versus hyphal
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4. 288 Journal of Biomolecular Screening 20(2)
growth. This was confirmed by LWR and MOS values,
which remained less than 1.5 independently of time points.
However, after 24 h, the Δefg1 grew as elongated yeast cells.
These elongated cells additionally strongly adhered to each
other, resulting in large clumps, which could resemble hyphal
growth. This complicated the MOS and LWR analysis. For
this reason, we focused on the analysis of 3 and 6 h. Moreover,
according to percentage of cellular viability, both mutant
strains are metabolically active and grow at these time points,
although Δefg1 does so to a slightly lower extent than Δedt1
(Fig. 3). This most likely stems from different growth rates of
the two mutant strains. In the stationary phase (after 24 h),
ATP amounts of the mutant strains were more equal again
and even exceeded those of the wild-type strain, presumably
due to an increased number of metabolically active yeast
cells as compared with hyphal growth. Hence, the mutant
strains can be used as references to screen for Y-H transition
inhibitory compounds.
Assays for the identification of morphotype inhibitors
have been described previously.13–15
These assays are
dependent on fluorescent reporter strains based on the pro-
moter of the hyphal wall protein HWP1, which is hypha
specific. The tag was introduced downstream of the pro-
moter either with green fluorescent protein (GFP) or beta-
galactosidase enzyme (lacZ).13–15
Methods based on
reporters, however, may also identify compounds that inter-
fere directly with GFP or beta-galactosidase rather than
influence filamentous growth. Moreover, it might be possi-
ble that upon activity of a potential compound, hyphal
growth is blocked and the promoters are still active, giving
rise to a false-negative signal.15
In addition, the incubation
time with 4 h is shorter than our analysis spanning from 3 h
to 6 h for the identification of switching blockers and up to
24 h for the identification of fungicidal compounds. Thus,
using the previously presented methods, the possibility
remains that effects of the compounds are only temporarily
Figure 1. Length/width ratio (LWR) and mean object shape (MOS) define cell morphology. The cells were incubated at 37 °C and
5% CO2
for 3 and 6 h (A, C, and B, D). C. albicans SC5314 in 0.5% DMSO served as hyphal growth control. The other samples
represent conditions in which yeast growth was prevalent. In the presence of farnesol (250 µM), C. albicans remained growing as yeast.
Thimerosal treatment is lethal to C. albicans, resulting in exclusively dead yeast-form cells. The two yeast-locked mutant stains Δedt1
and Δefg1 are unable to grow as hyphae and thus served as genetic controls of the approach. LWR and MOS values less than 1.5 (eq
1) are considered as growth in the yeast morphotype, meaning a distinct difference of cells in the hyphal morphotype (dotted line).
Data were analyzed using a one-way analysis of variance and Tukey’s multiple comparison tests. After 3 and 6 h, the conditions with
yeast growth were significantly different from the DMSO control (p ≤ 0.001). Furthermore, the Z′ factor for 6 h MOS and LWR is
≥0.5 (eq 3), which confirms the validity of the method.
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5. Stylianou et al. 289
or that the compounds are fungistatic at later time points, as
it is the case for farnesol.8
Our method is based on the type strain C. albicans
SC5314 and importantly is applicable to any other wild-
type strain from other fungal species. It is optimized for
automated liquid handling using small volumes and can be
carried out according to AFST guidelines. In this one-step
screening approach, the positive hits are detected by means
of LWR and MOS. An advantage of this microscopic assay
compared with microplate reader assays is that actual image
information from each calculation is stored and allows veri-
fication of different parameter measurements at later time
points. As a second step, we tested positive-hit compounds
from the primary screening for fungistatic/fungicidal activ-
ity by measuring cell viability via ATP levels. As demon-
strated by the use of yeast-locked mutant C. albicans strains,
this approach is suitable for distinguishing fungistatic or
fungicidal compounds from morphotype-switching inhibi-
tors (Fig. 4). The Z′ factor was introduced as a valuable tool
to validate the quality of screening assays.11
A Z′ factor
value between 0.5 and 1 defines the method as an excellent
assay, suitable and valid for high-throughput screening.
Notably, we found that the mean Z′ factor for LWR and
MOS at 6 h was 0.513, confirming that our method is a
highly suitable screening assay. All Z′ factor values were
calculated from at least three biological replicates. At 3 h,
the mean Z′ factor for LWR and MOS reached only a value
of 0.1. These Z′ factor values define a smaller separation
band at 3 h than at 6 h. Nevertheless, the trend is already
confirmed at 3 h.
In summary, we aimed to establish a high-throughput
screening method to find compounds that break the Y-H
switching. The identified compounds will have the potential
to disarm the pathogen without disturbing the cellular via-
bility, probably resulting in low selection pressure. A great
advantage of our method is that it may serve as blueprint for
screening with other polymorphic fungal pathogens,
because wild-type strains without genetic modifications are
applicable. Conclusively, our proposed method is a valu-
able tool for the identification of new and more efficient
antimycotics.
Acknowledgments
We would like to thank Steffen Rupp and Robert Wheeler for
kindly providing C. albicans strains.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
work was supported by grants to C.F.U. from the Swedish
Research Council VR-M (2011-2393), the Laboratory for
Molecular Medicine Sweden (MIMS), the Åke Wiberg Foundation
(3772734), and the Medial Faculty Umeå (316-886-10). M.S.
acknowledges financial support from the J.C. Kempes Memorial
Fund. Laboratories for Chemical Biology Umeå (LCBU) and
Figure 2. Microscopic images confirm quantification results
via length/width ratio and mean object shape. The cells were
incubated at 37 °C and 5% CO2
for 3 and 6 h (A, C, E, G, I
and B, D, F, H, J). C. albicans SC5314 in 0.5% DMSO served as
the hyphal growth control (A, B). The other samples represent
conditions in which yeast growth was prevalent, with 250 µM
farnesol (C, D) upon thimerosal treatment (E, F) and the two
yeast-locked mutant stains Δedt1 (G, H) and Δefg1 (I, J).The
pictures were captured from an ArrayScan microscope with a
10× objective lens, and the scale bar corresponds to 150 µm.
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6. 290 Journal of Biomolecular Screening 20(2)
Chemical Biology Consortium Sweden (CBCS) thank the Swedish
Research Council, the Swedish Governmental Agency for
Innovation Systems (VINNOVA), the Knut & Alice Wallenberg
foundation, the Kempe foundations, and the Carl Trygger founda-
tion for support. The funders had no role in the design or evalua-
tion of the research.
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