Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis
Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.
EUKARYOTIC CELL, June 2011, p. 803–819 Vol. 10, No. 6
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Calcineurin Controls Drug Tolerance, Hyphal Growth, and
Virulence in Candida dubliniensis
Emma L. Morrison,
Fitz Gerald S. Silao,
Ursela G. Bigol,
Fedelino F. Malbas, Jr.,
Jeniel E. Nett,
David R. Andes,
Norma V. Solis,
Scott G. Filler,
and Joseph Heitman
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina
; Aberdeen Fungal Group,
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom
; Department of
Microbiology and Parasitology, University of Perpetual Help-Dr. Jose G. Tamayo Medical University, Bin˜an, Laguna, Philippines
Environment and Biotechnology Division, Department of Science and Technology, Bicutan, Philippines
; Research Institute for
Tropical Medicine, Alabang, Philippines
; Departments of Medicine
and Medical Microbiology and Immunology,
University of Wisconsin, and William S. Middleton Memorial Veterans Hospital,
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California
David Geffen School of Medicine at UCLA, Los Angeles, California
Received 9 December 2010/Accepted 11 April 2011
Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and
frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C.
dubliniensis infection is common and constitutes a signiﬁcant therapeutic challenge. The calcineurin
inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal patho-
gens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin
is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins;
hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C.
albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that
calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation
in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin,
in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C.
dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection
model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore,
we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form
bioﬁlms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes
fungus-speciﬁc calcineurin inhibitors attractive candidates for combination therapy with azoles or echi-
nocandins against emerging C. dubliniensis infections.
Although Candida albicans is the most prevalent species
causing candidiasis, ⬎40% of Candida infections are now
caused by evolutionarily diverged non-albicans Candida spe-
cies (NACS). Candida dubliniensis, an emerging NACS that
occurs globally, was ﬁrst described as a separate species in 1995
(80), and its complete genome was recently sequenced (41). C.
dubliniensis is the closest relative of the important human fun-
gal pathogen C. albicans and commonly isolated from the oral
cavities of patients with AIDS or individuals who are human
immunodeﬁciency virus (HIV) positive and is occasionally
found in the oral microﬂora of healthy individuals (78). Clin-
ically, C. dubliniensis causes 2 to 7% of candidemia cases (40,
79), and it has been suggested that the gastrointestinal tract is
a source of the C. dubliniensis in candidemia patients (13).
Moreover, C. dubliniensis is now ranked as either the second or
third most frequently isolated Candida species from patients
with HIV/AIDS (6, 82). Interestingly, in addition to humans as
the source, C. dubliniensis can be isolated from nonhuman
sources, including ticks that parasitize seabirds (56) and the
excrement of seabirds (49). Most avian C. dubliniensis isolates
are genetically distinct from human isolates, but one avian
isolate (AV7) has been shown to be indistinguishable from a
human isolate by multilocus sequence typing (49), suggesting
that transmission may occur between birds and humans.
C. dubliniensis isolates are susceptible to azole antifungal
agents. However, C. dubliniensis can rapidly develop azole resis-
tance during clinical therapy (52, 64). Chunchanur et al. recently
reported that ⬃23% of C. dubliniensis isolates from HIV-infected
patients were resistant to ﬂuconazole (22). Moreover, ERG11
mutations in C. dubliniensis isolated from HIV-infected indi-
viduals contribute to decreased susceptibility to ﬂuconazole
(64). Thus, new therapies that involve novel or combination
drug treatments are needed. The calcineurin inhibitors tacroli-
mus (FK506) and cyclosporine A (CsA) target calcineurin
through the intracellular receptor FK506 binding protein 12
(FKBP12) or cyclophilin A (CyA), respectively. Calcineurin is
a eukaryotic calmodulin-dependent serine/threonine protein
phosphatase. It forms a heterodimer protein consisting of the
catalytic A (Cna1) and regulatory B (Cnb1) subunits, which are
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, Duke University Medical Center, Dur-
ham, NC 27710. Phone: (919) 684-2824. Fax: (919) 684-5458. E-mail:
† Supplemental material for this article may be found at http://ec
Published ahead of print on 29 April 2011.
highly conserved between yeasts and mammals (3). In response
to stress, the transcription factor Crz1 is dephosphorylated by
calcineurin and then migrates to the nucleus to regulate ex-
pression of genes encoding cell wall biosynthetic enzymes and
proteins involved in ion homeostasis (42, 72, 75, 76). Calcineu-
rin is required for azole and/or echinocandin tolerance in C.
albicans (61, 70, 84), C. neoformans (28, 45), and A. fumigatus
(74); thus, the combination of a calcineurin inhibitor with ei-
ther class of antifungal drug results in synergistic fungicidal
The ability to undergo dimorphic transitions is integral to
the virulence of C. albicans. The C. albicans ability to produce
yeast cells is critical for dissemination, whereas the ability to
form hyphae underlies survival and escape from macrophages
and the ability to penetrate and invade tissues (15). Mutants
locked in either the yeast (cph1 efg1) (47) or hyphal (tup1)
form (14) exhibit attenuated virulence in murine systemic in-
fection models. The role of the calcineurin pathway in hyphal
growth of C. albicans is unclear. Two groups, including our
own, found no role for calcineurin or Crz1 in hyphal growth (5,
62), while one group presented evidence interpreted to suggest
a role for calcineurin and Crz1 in hyphal growth on ﬁlament-
inducing media (42, 68). These differing results might be due to
different genetic backgrounds of the strains or experimental
protocols. C. dubliniensis is the only NACS capable of produc-
ing true hyphae, although morphogenesis is typically less ro-
bust than C. albicans on most ﬁlament-inducing media, which
could explain its attenuated virulence in a murine systemic
infection model compared with C. albicans (77). Thus, it is of
interest to investigate the roles of calcineurin in hyphal growth
and virulence in C. dubliniensis. In addition, C. albicans cal-
cineurin and Crz1 are required for tropic responses, a pheno-
type linked to hyphal growth. C. albicans Crz1 is involved in
thigmotropism and galvanotropism while calcineurin is in-
volved in galvanotropism, suggesting that tropic responses are
Crz1 dependent (9–11). In addition to hyphal growth, survival
in serum is essential for pathogenic Candida species to dissem-
inate and proliferate in the host. In C. albicans, calcineurin is
required for serum survival (5, 8, 68). However, in the basid-
iomycete C. neoformans, calcineurin is not required for growth
in serum and instead is required for growth at 37°C (59). Thus,
fungal pathogens employ calcineurin in divergent roles to es-
tablish infection, while the mammalian host employs calcineu-
rin as a defense against fungal infections via both innate im-
munity and adaptive immunity (37, 75).
In this study we investigate the roles of calcineurin in growth
and pathogenesis in C. dubliniensis. We show that calcineurin
is required for cell wall integrity, hyphal growth, serum sur-
vival, and virulence in C. dubliniensis, underscoring the impor-
tance of calcineurin as a global fungal virulence determinant
and potential drug target. Furthermore, we demonstrate that
calcineurin is required for azole or echinocandin tolerance,
suggesting a possible combination therapy in C. dubliniensis,
which frequently infects patients with HIV/AIDS.
MATERIALS AND METHODS
Yeast strains, media, and chemicals. Fungal strains used in this study are listed
in Table 1. The following media were used in this study: YPD (1% yeast extract,
2% peptone, 2% glucose) liquid medium and agar (2%) plates, spider medium
(10 g nutrient broth, 10 g mannitol,4gK
, 14 g Bacto agar in 1 liter
double-distilled water [ddH
O]; pH was adjusted to 7.2 with H
), serum agar
(50% serum, 2% agar), synthetic low-ammonium dextrose [SLAD; 1.7 g yeast
nitrogen base without amino acids and without ammonium sulfate, 20 g glucose,
, 20 g Bacto agar in 1 liter of ddH
O], and ﬁlament
agar (FA; 1.7 g yeast nitrogen base without amino acids and without ammonium
sulfate, 5 g glucose, 40 g Bacto agar in 1 liter ddH
O). YPD medium containing
100 g/ml nourseothricin was used to select transformants. The supplements
FK506 (Astellas Pharma Inc.), cyclosporine A (CsA; LC Laboratories), sodium
dodecyl sulfate (SDS; Fisher), fetal bovine serum (Invitrogen), calcoﬂuor white
(CFW; ﬂuorescent brightener 28; Sigma), Congo red (Sigma), ﬂuconazole (Bed-
ford Laboratories), posaconazole (Sequoia Research Products Ltd.), voricona-
zole (Sigma), caspofungin (Merck), micafungin (Astellas Pharma Inc.), and
anidulafungin (Pﬁzer Inc.) were added to the media at the concentrations indi-
Strain construction. Both alleles of the C. dubliniensis CNA1, CNB1, and
CRZ1 genes were disrupted with the SAT1 ﬂipper (66). For the CNA1 gene
disruption, approximately 1-kb 5⬘ (ampliﬁed with primers JC57/JC58; see Table
S1 in the supplemental material) and 3⬘ (ampliﬁed with primers JC59/JC60)
noncoding regions (NCRs) of the CNA1 open reading frame (ORF) were PCR
ampliﬁed from genomic DNA of the wild-type strain CD36. The 4.2-kb SAT1
ﬂipper sequence was ampliﬁed from plasmid pSFS2A (66) with primers JC17/
JC18. The three PCR products were treated with ExoSAP-IT (USB Corp.) to
remove contaminating primers and deoxynucleotide triphosphates (dNTPs) and
then combined in a 1:3:1 molar ratio (5⬘CNA1
generate the disruption allele by overlap PCR using ﬂanking primers JC61/JC62
(⬃100 bp closer to the CNA1 ORF compared with JC57/JC60, respectively,
reserving primers JC57/JC60 for further integration conﬁrmation), resulting in
an ⬃6-kb 5⬘CNA1
CNA1 disruption allele. The
ﬁrst allele of the CNA1 gene was disrupted in the wild-type strain CD36 by
transformation with 0.2 to 1 g of gel-puriﬁed disruption DNA by electropora-
tion (17). Two independent heterozygous nourseothricin-resistant mutants
(YC31 and YC29; Table 1) were obtained from two separate transformations.
Liquid YPM (1% yeast extract-2% peptone-2% maltose) medium was used to
drive expression of the FLP recombinase under the control of C. albicans MAL2
promoter (see Fig. S1 in the supplemental material). The SAT1 ﬂipper was
then excised, leaving an FLP recombination target (FRT), and resulted in
nourseothricin-sensitive CNA1/cna1 mutant strains (YC36 and YC73). The sec-
ond allele of the CNA1 gene was disrupted with the same overlap PCR allele,
resulting in nourseothricin-resistant homozygous cna1/cna1 mutants YC40 and
YC94 (Table 1). A similar approach was employed to disrupt the CNB1 and
CRZ1 genes, with ⬃0.7-kb 5⬘ and 3⬘ noncoding regions for homologous recom-
bination. To generate the ⬃5.4-kb cnb1 disruption allele, the overlap PCR DNA
(ampliﬁed with primers JC82/JC83), SAT1 ﬂipper (ampli
ﬁed with primers JC17/JC18), and 3⬘CNB1
(ampliﬁed with primers JC86/
JC87) were mixed in a 1:3:1 molar ratio and ampliﬁed with primers JC88/JC89
(⬃100 bp closer to the CNB1 ORF compared with JC82/JC87, respectively). Two
independent nourseothricin-resistant cnb1/cnb1 mutants (YC87 and YC96; Ta-
ble 1) derived from two separate transformations were obtained. To generate the
⬃5.4-kb crz1 disruption allele, 5⬘CRZ1
(ampliﬁed with primers JC100/
JC101), SAT1 ﬂipper (ampliﬁed with primers JC17/JC18), and 3⬘CRZ1
pliﬁed with primers JC102/JC103) were combined and ampliﬁed with primers
JC104/JC105 (⬃100 bp closer to the CRZ1 ORF compared with JC100/JC103,
respectively). Two independent nourseothricin-resistant crz1/crz1 mutants
(YC107 and YC108; Table 1) derived from two separate transformations were
obtained. These mutants were conﬁrmed by PCR (data not shown) and validated
by Southern blot analysis (Fig. S1).
The CRZ1 complementation construct was made by amplifying a 0.45-kb
fragment containing the 3⬘ NCR from CD36 genomic DNA with the JC320/
JC321 primers that introduced SacII and SacI sites. This fragment was cleaved
and ligated into the SacII-SacI-digested plasmid pGM175 (a gift from Gary
Moran), resulting in pYC389. The 2.8-kb fragment containing the 5⬘ NCR and
CRZ1 ORF ampliﬁed with the JC288/JC289 primers that introduced KpnI and
HindIII sites was cleaved and ligated into the KpnI-HindIII-digested pYC389,
resulting in pYC393. The 8-kb fragment containing the CRZ1 complementation
allele and SAT1 marker (see Fig. S2A in the supplemental material) was ampli-
ﬁed from pYC393 with the JC288/JC321 primers, and this fragment was used to
transform the nourseothricin-sensitive crz1/crz1 mutant (YC280, derived from
YC108; Table 1) to generate the CRZ1-complemented strain YC512 (Table 1).
PCR and Southern blot analyses (Fig. S2A) were used to conﬁrm the integration
of the CRZ1 complementation allele.
Southern blot analysis. Genomic DNA was isolated with the MasterPure yeast
DNA puriﬁcation kit (Epicentre Biotechnologies) from C. dubliniensis strains
grown either in liquid YPD culture or on YPD plates. Twenty micrograms of
804 CHEN ET AL. EUKARYOT.CELL
DNA was subjected to Southern blot analysis. The genomic DNAs of the cna1/
cna1, cnb1/cnb1, and crz1/crz1 mutants were digested with PpuMI, HpaI, and
EcoRV, respectively (see Fig. S1 in the supplemental material). PCR products
(ampliﬁed with primers JC57/JC58), 5⬘CNB1
pliﬁed with primers JC82/JC83), and 3⬘CRZ1
(ampliﬁed with primers JC102/
JC103) were used as probes. Radiolabeled probes were generated using the
Rediprime-it kit (Stratagene) and [␣-
P]dCTP (Easy Tides; Perkin-Elmer, Bos
ton, MA). Ultrahyb buffer (Ambion) was used for prehybridization at 42°C, and
hybridization and washing conditions were as described previously (17). Radio-
active signals were exposed onto the storage phosphor cassette and digitalized
with a Typhoon 9200 phosphorimager (Molecular Dynamics).
Spot growth assays. Cells were grown overnight at 30°C and washed twice with
O, and the optical density at 600 nm (OD
) was measured. Cells were
resuspended into an appropriate amount of ddH
O to achieve 1 OD/ml. Three
microliters of 5-fold serial dilutions (40 l of 1-OD/ml cells plus 160 l of ddH
as the ﬁrst dilution in a 96-well plate) from each strain was spotted with a
multichannel pipette onto solid media. The plates were then incubated at the
indicated temperatures for 48 h and photographed.
Quantitative determination of expression by real-time reverse transcription-
PCR (RT-PCR). Strains were grown overnight at 30°C and washed twice with
O. Cells were diluted to 0.2 OD/ml in YPD and incubated for3hat30°C.
Cells in log phase were then diluted to 0.2 OD/ml (10 ml) in YPD. Following 3 h
of incubation at 30°C/250 rpm, cells were pelleted at 3,000 rpm at ⫺4°C and
stored at ⫺80°C for further RNA extractions. The total RNAs were extracted
using the RNeasy minikit (Qiagen). RNA purity and integrity were determined
with a Nanodrop spectrophotometer and by gel electrophoresis, respectively. We
used DNase I (Turbo DNA-free; Ambion) to eliminate genomic DNA contam-
ination. Five hundred nanograms of DNA-free total RNAs was reverse tran-
scribed to cDNA by the Afﬁnity Script quantitative PCR (qPCR) cDNA synthesis
kit (Agilent). PCR mixtures of 25 l included 5 ng cDNA (in 10 l), 12.5 lof
2⫻ qPCR master mix (Brilliant SYBR green kit; Agilent), 0.5 lof5M forward
primer, 0.5 lof5M reverse primer, 1.125 l of nuclease-free H
O, and 0.375
l of ROX dye. Quantitative PCR conditions were the following: 95°C for 10 min
(denaturation); 95°C for 15 s and 60°C for 1 min (40 times, cycling stage); and
95°C for 15 s, 60°C for 1 min, and 95°C for 15 s (melting curve). Primers for
probes were designed using Primer3 (http://frodo.wi.mit.edu/primer3/) and are
TABLE 1. C. dubliniensis, C. albicans, and C. neoformans strains used in this study
Strain Genotype Background Reference
CD36 Prototrophic wild type Clinical isolate 80
YC31 CNA1/cna1::SAT1-FLP CD36 This study
YC36 CNA1/cna1::FRT YC31 This study
cna1::FRT/cna1::SAT1-FLP YC36 This study
YC29 CNA1/cna1::SAT1-FLP CD36 This study
YC73 CNA1/cna1::FRT YC29 This study
cna1::FRT/cna1::SAT1-FLP YC73 This study
YC47 CNB1/cnb1::SAT1-FLP CD36 This study
YC69 CNB1/cnb1::FRT YC47 This study
cnb1::FRT/cnb1::SAT1-FLP YC69 This study
YC41 CNB1/cnb1::SAT1-FLP CD36 This study
YC82 CNB1/cnb1::FRT YC41 This study
cnb1::FRT/cnb1::SAT1-FLP YC82 This study
YC81 CRZ1/crz1::SAT1-FLP CD36 This study
YC102 CRZ1/crz1::FRT YC81 This study
crz1::SAT1-FLP/crz1::SAT1-FLP YC102 This study
YC80 CRZ1/crz1::SAT1-FLP CD36 This study
YC100 CRZ1/crz1::FRT YC80 This study
crz1::FRT/crz1::SAT1-FLP YC100 This study
YC280 crz1::FRT/crz1::FRT YC108 This study
YC512 crz1::FRT/crz1::FRT ⫹ CRZ1 YC280 This study
SC5314 Prototrophic wild type Clinical isolate 36
SCCMP1M4 cna1::FRT/cna1::FRT SC5314 5
SCCMP1MK2 cna1::FRT/cna1::FRT ⫹ CNA1 SCCMP1M4 5
DAY185 ura3/ura3 his1::hisG/his1::hisG::HIS1
JRB64 ura3/ura3 arg4/arg4 his1/his1 cnb1::URA3/cnb1::UAU1 ⫹ HIS1 BWP17 8
MCC85 ura3/ura3 his1::hisG::CNB1-HIS1/his1::hisG
OCC1.1 ura3/ura3 his1::hisG::HIS1/his1::hisG arg4::hisG/arg4::hisG
OCC7 ura3/ura3 his1::hisG::CRZ1-HIS1/his1::hisG
CAF2-1 ura3/URA3 SC5314 32
DSY2091 cna1::hisG/cna1::hisG::URA3::hisG CAF2-1 68
DSY2115 cna1::hisG/cna1::hisG LEU2::CNA1::URA3 CAF2-1 68
DSY2195 crz1::hisG/crz1::hisG::URA3::hisG CAF2-1 42
MKY268 crz1::hisG/crz1::hisG LEU2::CRZ1/URA3 CAF2-1 42
H99 Prototrophic wild type Clinical isolate 65
KK1 cna1::NAT H99 44
Two independent cna1/cna1 mutants.
Two independent cnb1/cnb1 mutants.
Two independent crz1/crz1 mutants.
VOL. 10, 2011 ROLES OF CALCINEURIN PATHWAY IN C. DUBLINIENSIS 805
listed in Table S1 in the supplemental material. An ABI Prism 7900HT machine
and StepOne software v2.1 (Applied Biosystems) were used to determine thresh-
old cycle (⌬⌬CT) and relative quantity (RQ). The bar graphs of ACT1-normal-
ized RQ compared with the wild type (CD36) were created with Prism 5.03.
Disk diffusion assays. Cells were grown overnight at 30°C and diluted to 1
OD/ml, and then 100 l (0.1 OD) was spread onto YPD in the absence or
presence of FK506 (1 g/ml) or CsA (100 g/ml). After 10 min, sterile disks were
placed onto the surface of the agar. Ten microliters of 0.1-g/l ﬂuconazole (1
g total) or H
O (control) was spotted onto the sterile disk after placement on
the medium. The plates were then incubated at 30°C for 24 h and photographed.
Time-kill curve for strains exposed to ﬂuconazole. Cells were grown overnight
at 30°C and washed twice with ddH
O. Cells were separated by sonication
(Branson sonicator 250) at a constant of 2 for 10 s and counted with a hemocy-
tometer. Cells (5 ⫻ 10
) were added to 5 ml of fresh YPD medium to achieve 10
cells/ml in the absence or presence of ﬂuconazole (10 g/ml). Cells were cultured
at 30°C with shaking at 250 rpm. The cells surviving after 0, 3, 6, 9, and 24 h were
serially diluted onto YPD medium, and CFU were counted after 48 h of incu-
bation. The experiments were performed in triplicate, and data were plotted
using Prism 5.03.
Germ tube formation assays. Cells were grown overnight and washed twice
O. Cells were diluted to 1 OD/ml and sonicated to separate slightly
clumped cells. Two microliters of cells was added to microtiter wells preﬁlled
with 98 l of serum or spider medium, resulting in 0.002 OD (⬃4 ⫻ 10
each well. Strains were conﬁrmed to have no germ tubes at 0 h and were
incubated at 37°C for the indicated times. The percentage of germ tube forma-
tion was counted using the following formula [(germ tube cells)/(germ tube ⫹
yeast cells)] ⫻ 100%. At least 200 cells were counted in each experiment of three
Thigmotropism assays. Poly-
L-lysine-coated quartz slides featuring ridges of
0.79 m ⫾ 40 nm and a pitch of 25 m (Kelvin Nanotechnology, Glasgow,
United Kingdom) (11) were prepared by treatment with UV-ozone for 2 min
followed by coating with 0.01% (wt/vol) poly-
L-lysine (150,000 to 333,000 M
Sigma, United Kingdom) for 30 min. Slides were rinsed with ddH
O and left
overnight to dry in a sterile petri dish. Yeast cells were grown overnight in 5 ml
YPD with shaking at 200 rpm. A volume of 7.5 l was added to 10 ml ddH
After vortexing, the suspension was poured over a quartz slide and cells were
allowed to adhere at room temperature for 30 min. Slides were lightly rinsed with
O to remove unadhered cells and placed in 20 ml prewarmed 20% (vol/vol)
newborn calf serum containing 2% (wt/vol) glucose at 37°C for6htoinduce
hyphae. The number of hyphae reorienting on contact with a ridge was deter-
mined by light microscopy, and tip reorientations were expressed as a percentage
of the total observed interactions. At least 100 interactions were observed per
strain in each experiment, and results were reported as the mean value from
three independent experiments ⫾ standard deviation (SD).
Murine systemic infection model. Five- to 6-week-old male CD1 mice from
Jackson Laboratories (n ⫽ 15 for each group, except n ⫽ 10 for C. albicans) were
used in this study. C. dubliniensis and C. albicans strains were grown in 5 ml YPD
overnight at 30°C with shaking at 250 rpm. Cultures were washed twice with 10
ml of phosphate-buffered saline (PBS), and the cells were then resuspended in 2
ml of PBS. Modestly clumped cells were dispersed by sonication. Cells were
counted with a hemocytometer and resuspended in an appropriate amount of
PBS to obtain an infection inoculum of 2.5 ⫻ 10
cells/ml. Two hundred micro
liters (5 ⫻ 10
cells) was used to infect mice by lateral tail vein injection. The
course of infection was monitored for up to 42 days. The survival of mice was
monitored twice daily, and moribund mice (unable to eat or drink, body weight
reduced by ⬎30%, severely tilted head, or hunched) were euthanized with CO
All experimental procedures were carried out according to NIH guidelines and
Duke University Institutional Animal Care and Use Committee (IACUC) pro-
tocols for the ethical treatment of animals. Appropriate dilutions of the cells
were plated onto YPD and incubated at 30°C for 48 h to conﬁrm cell viability.
To determine fungal burden, the left kidney of C. dubliniensis-infected mice
(n ⫽ 5 for each strain, except n ⫽ 4 for strain YC107 due to a death immediately
following injection) was dissected at day 7. The organs were weighed, transferred
to a 15-ml Falcon tube ﬁlled with 5 ml PBS, and homogenized for 10 s at 19,000
rpm/min (IKA T25; Cole-Parmer). Tissue homogenates were serially diluted,
and 100 l was plated onto a YPD plate. The plates were incubated at 30°C for
48 h to determine CFU per gram of kidney. The identity of organ-recovered
colonies was conﬁrmed by PCR and by growth or no growth on YPD medium
containing 0.01% SDS. For histopathological analysis, kidneys were excised at
day 14, ﬁxed in 10% phosphate-buffered formalin (Fisher), and Gomori methe-
namine silver (GMS) and hematoxylin and eosin (H&E) stainings were per-
formed by the Department of Pathology at Duke University. After slide prepa-
ration, each sample was examined thoroughly by microscopy for analysis of
Candida colonization (GMS) and tissue necrosis (H&E). Images were captured
using an Olympus Vanox microscope (PhotoPath; Duke University Medical
Murine ocular infection model. Cells were grown in YPD broth overnight at
37°C. Cultures were pelleted by centrifugation (10,000 rpm for 15 min) and
washed three times with sterile PBS (pH 7.4). Cells were suspended and diluted
in sterile PBS to yield a fungal concentration of 10
cells/5 l. Concentration was
determined by using spectrophotometer optical density reading at a 600-nm
wavelength and multiplying it with a conversion factor of 1 OD
, equivalent to
3 ⫻ 10
cells/ml. Inoculum concentration was veriﬁed by plating on YPD for 48 h
For murine ocular infection, outbred ICR mice (Research Institute for Trop-
ical Medicine, Alabang, Philippines) around 6 to 8 weeks of age (20 to 25 g) were
used in the experiment in accordance with the ARVO statement for the Use of
Animals in Ophthalmic and Vision Research. An experimental keratomycosis
protocol described previously (89) was used for C. dubliniensis with minor mod-
iﬁcations and was approved by the University of Perpetual Help Institutional
Review Board. Mice were maintained in comfortable cages with a constant
supply of food and water, and the cages were periodically sanitized with Sterilium
to minimize potential other infections during the course of the observations.
Mice were immunocompromised by intraperitoneal injection of cyclophos-
phamide (180 to 200 mg/kg body weight; Sigma-Aldrich) predissolved in sterile
0.9% NaCl 5 days, 3 days, and 1 day before the inoculation. Prior to inoculation,
mice were anesthetized by intramuscular injection of Zoletil 50 (15 mg/kg body
weight; Virac, Australia) followed by topical application of proparacaine hydro-
chloride ophthalmic solution (Alcaine; Alcon-Couvreur, Belgium) to the eyes.
Once animals were anesthetized, the right eye was superﬁcially scariﬁed in a grid
pattern with a sterile 25-gauge hypodermic needle. Five microliters of Candida
cells) was placed into each eye. Inoculum was distributed uniformly
by rubbing the eye with the eyelid. A mock-infection experiment was performed
using sterile PBS as control. Disease severity of fungal keratitis was assessed for
8 days with the aid of a dissecting microscope based on the procedure described
previously (89). In this procedure, corneal involvement was assessed and scored
according to three parameters, namely, (i) area of opacity, (ii) density of opacity,
and (iii) surface regularity. A grade of 0 to 4 was assigned on each of these
criteria to yield a maximum score of 12.
Wax moth infection studies. Wax moths (Galleria mellonella) of the ﬁnal-instar
larval stage (⬃0.3 g) from Vanderhorst (Ohio) were used (10 per strain) within
7 days from the day of shipment. The larval infection protocol was adapted from
previously described methods for C. neoformans (53) with minor modiﬁcations.
Each larva was infected with 10
C. dubliniensis or C. albicans cells in 5 l PBS
by injection into the last pseudopod and incubated at 24°C in a petri dish with
wood shavings. Larvae showing signs of severe morbidity, such as change of body
color and no response to touch, were sacriﬁced by cold treatment at ⫺20°C. The
number of surviving wax moths was monitored and recorded daily.
Epithelial cell interactions. The extent of damage to oral epithelial cells
caused by C. dubliniensis compared to C. albicans was determined by a minor
modiﬁcation of our previous method (63). Brieﬂy, the FaDu oral epithelial cell
line (American Type Culture Collection) was grown to 95% conﬂuence in a
24-well tissue culture plate and loaded with
Cr overnight. The following day,
the cells were rinsed extensively to remove the unincorporated tracer. Next, they
were infected with either 2 ⫻ 10
yeast cells of C. dubliniensis CD36 or 5 ⫻ 10
yeast cells of C. albicans SC5314 in 1 ml of RPMI 1640 medium. After 2, 4, and
6 h, an aliquot of the medium above the cells was withdrawn for determination
Cr content. Control wells containing uninfected epithelial cells were pro
cessed in parallel to determine the spontaneous release of
Cr. At the end of the
experiment, the cells were lysed with NaOH and the wells were rinsed with
RadiacWash (Atomic Products). The lysate and rinses were collected, and their
Cr content was determined. Each organism was tested in triplicate wells.
Differential interference contrast microscopy was used to examine the mor-
phology of the organisms when they were in contact with epithelial cells. FaDu
oral epithelial cells were grown to 95% conﬂuence on ﬁbronectin-coated glass
coverslips (12-mm diameter) in 24-well tissue culture plates. The epithelial cells
were infected with either 10
cells of C. dubliniensis CD36 or 5 ⫻ 10
cells of C.
albicans SC5314 in 1 ml of RPMI 1640 medium for 2, 4, or 6 h. Next, the
coverslips were rinsed once with PBS and then ﬁxed for 15 min with 3% para-
formaldehyde in PBS. They were then rinsed in PBS, mounted inverted on
microscope slides, and imaged by confocal microscopy.
Rat denture bioﬁlm model. Denture appliances were placed in speciﬁc-patho-
gen-free male Sprague-Dawley rats weighing ⬃350 g (Harlan Sprague-Dawley,
Indianapolis, IN) as previously described (54). Animals were anesthetized and
immunosuppressed with a single dose of cortisone (200 mg/kg subcutaneously
[SQ]) on the day of infection and received ampicillin-sulbactam, 100 mg/kg twice
806 CHEN ET AL. EUKARYOT.CELL
a day (BID), during the course of the experiment. A 32-gauge stainless steel
Babcock orthodontic wire (Miltex) was threaded across the hard palate and
secured between cheek teeth (54). Teeth were etched with Uni-Etch 32% semi-
gel etchant with benzalkonium chloride (Bisco, Inc.). A metal spatula was placed
over the hard palate to create a space for Candida inoculation. Cold cure acrylic
temporary crown and bridge material (HP MaxiTemp; Henry Schein) was ap-
plied over cheek teeth and wire and was allowed to solidify for 5 min. After
removal of the spatula, the hard palate beneath the acrylic device was inoculated
with Candida at 10
cells/ml (0.1 ml). Animals were sacriﬁced after 48 h of
denture placement, and devices were processed for scanning electron microscopy
(SEM) as previously described (2, 54). Brieﬂy, devices were washed with PBS and
placed in ﬁxative (1% [vol/vol] glutaraldehyde and 4% [vol/vol] formaldehyde in
PBS) overnight. The samples were rinsed with PBS, treated in 1% osmium
tetroxide for 30 min, and rinsed with PBS. The samples were then dehydrated in
a series of ethanol washes, and ﬁnal desiccation was accomplished by critical-
point drying (Tousimis, Rockville, MD). Specimens were mounted on aluminum
stubs and sputter coated with gold. Dentures were imaged on a JEOL 6100 at 10
kV. The images were processed for display using Adobe Photoshop.
Animals were maintained in accordance with the American Association for
Accreditation of Laboratory Animal Care (AAALAC) criteria, and all studies
were approved by the Institutional Animal Care and Use Committee (IACUC).
Statistical analysis. Statistical analysis was conducted using Prism 5.03 soft-
ware (GraphPad, La Jolla, CA), with the exception that SPSS software was used
to analyze thigmotropic responses (Dunnett’s t test). For the mouse and Galleria
larval infection studies, Kaplan-Meier survival curves were generated and the
log-rank (Mantel-Cox) test was employed to compare signiﬁcance. The signiﬁ-
cance of differences in fungal burden, germ tube formation, and real-time RT-
PCR was determined using one-way analysis of variance (ANOVA) and Dun-
nett’s multiple comparison tests. The signiﬁcance of the capacity of Candida
species to cause damage to oral epithelial cells and murine corneas was deter-
mined by unpaired t test and Student’s t test, respectively. A P value of ⬍0.05 was
Calcineurin mutation confers cell wall integrity defects in
C. dubliniensis. The newest class of antifungal drugs in clinical
use, the echinocandins, target fungal cell wall synthesis. There-
fore, there is increased interest in the study of Candida cell
wall integrity (19, 26, 87). Recently, Jackson et al. reported that
the genome sequences of C. dubliniensis and C. albicans are
highly conserved with considerable synteny, with the exception
of 168 species-speciﬁc genes which included cell wall-related
secreted aspartyl protease and agglutinin-like protein families
(41). Calcineurin is required for cell wall integrity in C. albi-
cans (26, 68) and A. fumigatus (73), but it is not known if
calcineurin has an analogous role in C. dubliniensis. The C.
dubliniensis orthologs of C. albicans CNA1/CMP1 and CNB1
and the calcineurin target CRZ1 genes were identiﬁed by re-
ciprocal BLAST searches between the two species and in all
cases identiﬁed a reciprocal best BLAST hit ortholog as the C.
dubliniensis CNA1 (CD36_00650), CNB1 (CD36_54760), and
CRZ1 (CD36_85720) genes. C. dubliniensis Cna1, Cnb1, and
Crz1 share 91%, 100%, and 81% identity, respectively, over
the full-length proteins with their corresponding C. albicans
orthologs. Calcineurin A (Cna1) has the conserved calcineurin
B binding, calmodulin-binding, and autoinhibitory regions.
Calcineurin B (Cnb1) has four EF-hand Ca
while Crz1 shares zinc ﬁnger domains with the respective
ortholog in C. albicans. Two independent calcineurin and
crz1/crz1 mutants were generated using the SAT1 ﬂipper
cassette and conﬁrmed by PCR and Southern blot analysis.
Real-time RT-PCR analysis conﬁrmed loss of expression of
the CNA1, CNB1,orCRZ1 gene in the respective null mu-
tant strains (Fig. 1A).
The cell wall integrity of the C. dubliniensis calcineurin
(cna1/cna1 and cnb1/cnb1) and crz1/crz1 mutants was assayed
by growing them in the presence of SDS, a reagent which
compromises cell membrane/wall integrity; calcoﬂuor white
(CFW), which destabilizes chitin polymerization; and Congo
FIG. 1. Calcineurin mutations result in defective cell wall integrity in C. dubliniensis. (A) Expression of the calcineurin and CRZ1 genes in the
wild-type and mutant strains was quantiﬁed by real-time PCR. The fold changes in transcription of CNA1, CNB1, and CRZ1 were normalized to
the endogenous control ACT1. The data are represented as means ⫾ SDs of triplicate measurements. One representative graph is shown from
three independent experiments. Asterisks indicate P ⬍ 0.0001 compared with the wild type. (B) Cells were grown overnight in YPD at 30°C, 5-fold
serially diluted, and spotted onto YPD medium containing sodium dodecyl sulfate (SDS), calcoﬂuor white (CFW), or Congo red (CR) and
incubated at 30°C for 48 h. WT, wild type.
OL. 10, 2011 ROLES OF CALCINEURIN PATHWAY IN C. DUBLINIENSIS 807
red, which intercalates between glucan polymers (30, 67, 83).
Similarly to C. albicans (26, 42, 62, 68), C. dubliniensis calcineu-
rin mutants exhibited hypersensitivity to SDS, while crz1/crz1
mutants exhibited a phenotype intermediate between the wild
type and calcineurin mutants (Fig. 1B), suggesting that cell
membrane/wall integrity controlled by calcineurin is at least in
part Crz1 dependent in C. dubliniensis. The slower growth of
these mutants on YPD medium containing SDS compared
with the wild type was not attributable to defects in thermal
stress tolerance (see Fig. S3A in the supplemental material) or
growth rate (Fig. S3B).
C. dubliniensis calcineurin, but not crz1/crz1, mutants were
hypersensitive to CFW (Fig. 1B), suggesting an essential role
of calcineurin in cell wall integrity. Interestingly, C. dubliniensis
calcineurin mutants were hypersensitive to Congo red, while
C. albicans calcineurin mutants exhibited wild-type growth
(Fig. 1B; up to 50 g/ml). This indicates that calcineurin plays
a greater role in cell wall integrity in C. dubliniensis than in C.
albicans. To examine if hypersensitivity to CFW and Congo red
of the C. dubliniensis calcineurin mutants was due to affected
chitin synthesis, we exposed the cells to nikkomycin Z, a chitin
synthase inhibitor (90). None of the mutants in either species
was hypersensitive to nikkomycin Z (1 to 10 g/ml) (data not
shown), in contrast to a calcineurin mutant (cnaA)ofA. fu-
migatus (33). This suggests a distinct role of calcineurin gov-
erning chitin synthesis in Candida and Aspergillus.
C. dubliniensis calcineurin mutants exhibit echinocandin
hypersusceptibility. Echinocandins (caspofungin, micafungin,
and anidulafungin) are a new class of antifungal drugs that
noncompetitively inhibit the cell wall biosynthetic enzyme
␤-1,3-glucan synthase, an essential enzyme in fungal patho-
gens. Kofteridis et al. recently reported that ⬃1% (7/650) of
Candida isolates from cancer patients with candidiasis are
caspofungin resistant (43). Resistance has also been reported
in other Candida clinical isolates (86), suggesting a need for
alternative therapy for invasive fungal infections. Singh et al.
demonstrated that calcineurin inhibitors (FK506 and CsA) ex-
hibit fungicidal activity with micafungin (at a nonfungicidal
concentration) against C. albicans (70), indicative of a poten-
tial combination therapy. However, synergism between cal-
cineurin inhibitors and echinocandins has not yet been inves-
tigated in C. dubliniensis.
Here, we report that, similar to C. albicans calcineurin mu-
tants, C. dubliniensis cna1/cna1 and cnb1/cnb1 mutants are
hypersusceptible to caspofungin, micafungin, and anidulafun-
gin compared with the wild type (Fig. 2). Interestingly, C.
dubliniensis crz1/crz1 mutants exhibited differential responses
to echinocandins. C. dubliniensis crz1/crz1 mutants showed an
intermediate hypersusceptibility to micafungin compared with
the wild type and calcineurin mutants, suggesting that mica-
fungin tolerance is mediated by Crz1. However, caspofungin
and anidulafungin did not affect the growth of crz1/crz1 mu-
tants (Fig. 2). To test if calcineurin is required for resistance to
caspofungin, we determined the MIC for the C. dubliniensis
calcineurin and crz1/crz1 mutants using Etest concentration-
gradient diffusion assays. The MIC for C. dubliniensis cna1/
cna1 and cnb1/cnb1 mutants was 0.016 g/ml (clear inhibition
zone) compared with 0.064 g/ml (turbid inhibition zone) for
the wild type, suggesting that calcineurin plays a role in caspo-
fungin resistance in C. dubliniensis (Table 2). The MIC for the
C. dubliniensis crz1/crz1 mutant (0.094 g/ml) was not signiﬁ-
cantly different from the wild type (Table 2). As a control
experiment, FK506 decreased caspofungin resistance in both
the wild type and the crz1/crz1 mutant but not in the calcineu-
rin mutants (Table 2), conﬁrming that resistance to caspofun-
gin in C. dubliniensis is orchestrated by calcineurin signaling.
Fluconazole tolerance is governed by calcineurin and Crz1
in C. dubliniensis. Azole-resistant C. dubliniensis is frequently
isolated from the oral cavities of HIV/AIDS patients (6, 82). A
calcineurin inhibitor and ﬂuconazole exhibited synergistic fun-
gicidal activity against C. albicans (26) and C. neoformans (28).
To test our hypothesis that calcineurin is required for ﬂucona-
zole tolerance in C. dubliniensis, we used spot, disk diffusion,
and time-killing curve assays. C. dubliniensis cna1/cna1 and
cnb1/cnb1 mutants were hypersusceptible to ﬂuconazole while
crz1/crz1 mutants exhibited susceptibility intermediate between
the wild type and the calcineurin mutants (Fig. 3A), suggesting
that other regulators control ﬂuconazole tolerance in addition
to Crz1. This is similar to C. albicans calcineurin and crz1/crz1
mutants, suggesting that azole tolerance governed by the cal-
cineurin pathway has been conserved during evolution of the
two Candida species. By disk diffusion assays, we found that
pharmacological inhibition of calcineurin phenocopies cal-
cineurin deletion while crz1/crz1 mutants exhibit an interme-
FIG. 2. Calcineurin mutations enhance fungicidal activity of echinocandins in C. dubliniensis. Cells were grown overnight in YPD at 30°C, 5-fold
serially diluted, and spotted onto YPD medium containing caspofungin (CS), micafungin (MF), or anidulafungin (AN) at the concentrations
indicated. The plates were incubated at 30°C for 48 h and photographed.
808 CHEN ET AL. E
diate effect between the wild type and calcineurin mutants in
C. dubliniensis (Fig. 3B). This strongly suggests that ﬂucona-
zole tolerance is controlled by other calcineurin downstream
targets in addition to the Crz1 transcription factor.
Time-killing curve assays showed that C. dubliniensis cna1/
cna1 and cnb1/cnb1 mutants initially (3 h) proliferated in the
presence of ﬂuconazole (10 g/ml) but that survival was dra-
matically decreased over 24 h compared with the wild type
(P ⬍ 0.0001; Fig. 3C). C. dubliniensis crz1/crz1 mutants exhib-
ited initial growth, but the growth rate dropped signiﬁcantly at
24h(P ⬍ 0.05; Fig. 3C). The synergistic fungicidal effects of
ﬂuconazole are therefore strongly linked to the loss of cal-
cineurin activity and are partially mediated by the transcription
factor Crz1. Calcineurin mutants also exhibited hypersuscep-
tibility to the new-generation azoles, posaconazole and vori-
conazole, in C. dubliniensis and C. albicans, while crz1/crz1
mutants showed differential susceptibility (see Fig. S4 in the
supplemental material). This suggests that azole tolerance is at
least in part mediated by the transcription factor Crz1. How-
ever, C. dubliniensis Crz2 (CD36_32610, encoding a putative
transcriptional regulator) does not play a role in azole toler-
ance because crz2/crz2 and crz2/crz2 crz1/crz1 mutants did not
exhibit hypersusceptibility compared with the wild type and
crz1/crz1 mutants, respectively (data not shown).
Calcineurin and Crz1 control cation homeostasis in C. dub-
liniensis. The roles of calcineurin and Crz1 in Ca
cation homeostasis have been elucidated in C. albicans
(18, 68, 69). Sanglard et al. showed that C. albicans cna1/cna1
mutants are hypersensitive to Ca
(1.5 M) (68) while
crz1/crz1 mutants are hypersensitive to divalent Ca
(42, 69). In other fungal pathogens, including C. neofor
mans (59), A. fumigatus (73), and Magnaporthe oryzae (21),
calcineurin is required for Ca
ion homeostasis. Our labora
tory previously showed that calcineurin is required for C. albi-
cans to survive Ca
exposure in serum and thereby for viru
lence (7, 8). These observations suggest a general role for
calcineurin in controlling Ca
homeostasis. Recently, Enjal
bert et al. showed that C. dubliniensis is Na
compared with C. albicans (31). Thus, it is of interest to inves-
tigate the potential roles of calcineurin in Na
homeostasis in C. dubliniensis.
Here, we demonstrate that, similarly to C. albicans, C. dub-
liniensis calcineurin (Cna1 and Cnb1) and Crz1 are essential
for growth in response to Ca
stress (Fig. 4
A). Surprisingly, at
elevated temperature (ⱖ30°C), crz1/crz1 mutants are hyper-
sensitive to Ca
compared with the calcineurin mutants in
two closely related species (see Fig. S5 in the supplemental
material). Interestingly, crz1/crz1 mutants exhibit intermediate
sensitivity phenotype compared with wild type and cal
cineurin mutants at 24°C in both C. dubliniensis and C. albicans
(Fig. S5). C. dubliniensis cna1/cna1 and cnb1/cnb1 mutants are
also hypersensitive to Mn
stress (Fig. 4A), whereas crz1/crz1
mutants exhibit intermediate sensitivity between the cal-
cineurin mutants and wild type, indicating that another reg-
ulator(s) in addition to Crz1 contributes to Mn
stasis. Interestingly, C. dubliniensis calcineurin mutants are
hypersensitive to Na
(1 M), while C. albicans calcineurin
mutants do not exhibit signiﬁcant differences compared with
their wild-type counterparts (Fig. 4A). In fact, C. albicans
calcineurin mutants are hypersensitive to a high Na
centration (2 M; data not shown). Thus, the difference in
roles of calcineurin in Na
ion homeostasis between two
closely related species (Fig. 4A) is due to the differential
sensitivity between the species and not to differences in
calcineurin activity in the two species.
The mechanisms of C. dubliniensis calcineurin and crz1/
crz1 mutants’ hypersensitivity to mono- or divalent cations
might involve defects in cation efﬂux systems, resulting in
cation accumulation in the cytosol. We show here that tran-
scription of PMC1 (encoding a vacuolar Ca
CD36_81200) is regulated by calcineurin and Crz1 in C.
dubliniensis (Fig. 4B, P ⬍ 0.001), indicating a mechanism by
likely accumulates in the cytosol of calcineurin
or crz1/crz1 mutants. The transcription of PMC1 was also
shown to be regulated by calcineurin and Crz1 in C. albicans
(42, 68) and in the rice blast pathogen M. oryzae (20). How-
ever, the transcription of PMR1 (encoding a Golgi Ca
transporter, CD36_70530), CCH1 (encoding a volt
channel, CD36_01040), and MID1
(encoding a Ca
channel, CD36_53710) was not controlled
by calcineurin or Crz1 (Fig. 4B) in C. dubliniensis.
Calcineurin but not Crz1 is required for serum survival in
C. dubliniensis. An essential role for calcineurin, but not Crz1,
in serum survival has been demonstrated in C. albicans (8), and
calcium stress in serum has been elucidated to be the cause of
lethality of calcineurin mutants (7). An inability to survive in
serum explains, at least in part, why C. albicans calcineurin
mutants exhibit attenuated virulence in a murine systemic in-
TABLE 2. Calcineurin is required for caspofungin resistance in C. dubliniensis
No FK506 FK506 (1 g/ml)
(MIC or range; g/ml)
(MIC or range; g/ml)
CD36 (wild type) 0.064 Turbid 0.016 Clear
cna1/cna1 (YC40) 0.016 Clear 0.016 Clear
cna1/cna1 (YC94) 0.016 Clear 0.012–0.016 Clear
cnb1/cnb1 (YC87) 0.016 Clear 0.016–0.023 Clear
cnb1/cnb1 (YC96) 0.016 Clear 0.016 Clear
crz1/crz1 (YC107) 0.094 Turbid 0.016 Clear
crz1/crz1 (YC108) 0.094 Turbid 0.016–0.023 Clear
Cells were grown overnight at 30°C and washed twice with ddH
O. Then 0.5 OD (in 500 l) of cells was spread on RPMI 1640 medium (Remel; R04067) in the
absence or presence of FK506 (1 g/ml). After 10 min, the Etest caspofungin strip (bioMérieux Corp.) was transferred to the surface of the medium. The MIC was
read after 24 h of incubation at 35°C according to the manufacturer’s instructions.
VOL. 10, 2011 ROLES OF CALCINEURIN PATHWAY IN C. DUBLINIENSIS 809
fection model but not in pulmonary or vaginal infection models
(5), indicative of niche-speciﬁc roles of calcineurin. However,
the roles of calcineurin and Crz1 have not yet been investi-
gated in the less virulent species C. dubliniensis. Here, we
demonstrate that, similar but not identical to C. albicans cal-
cineurin mutants, C. dubliniensis cna1/cna1 and cnb1/cnb1 mu-
tants were hypersensitive to serum but were in general less
sensitive than C. albicans calcineurin mutants (Fig. 5A), sug-
gesting an evolutionary divergence between two closely related
species. By quantitative measurements, we found that the
survival percentage of C. dubliniensis cna1/cna1 mutants
(0.354% ⫾ 0.125%) was 24-fold higher than C. albicans cna1/
cna1 mutants (0.015% ⫾ 0.006%) (P ⫽ 0.0094) on a 50%
serum agar plate (data not shown). However, Crz1 is not re-
quired for serum survival in either Candida species (Fig. 5 and
data not shown). We further characterized germ tube forma-
tion of the wild type and mutants in 100% serum. Similar to the
C. albicans calcineurin mutant, the C. dubliniensis calcineurin
mutants exhibit germ tube formation defects at 2 h and further
growth was inhibited over 24 h (Fig. 5B). However, C. dublini-
ensis Crz1 is not required for germ tube formation in liquid
100% serum (Fig. 5B). Taken together, calcineurin, but not
Crz1, is required for serum survival and germ tube formation
in 100% serum in C. dubliniensis.
FIG. 3. Calcineurin is required for ﬂuconazole tolerance in C. dubliniensis. (A) Cells were grown overnight in YPD at 30°C, 5-fold serially
diluted, and spotted onto YPD medium ⫾ ﬂuconazole (FL). The plates were incubated at 30°C for 48 h. (B) Disk diffusion assays were used to
determine ﬂuconazole susceptibility of wild-type and mutant strains. Cells were grown overnight at 30°C, and 0.1 OD
(in 100 l) was spread on
the surface of YPD medium ⫾ FK506 or CsA at the concentrations indicated. A disk was placed on the surface of the medium, and ﬂuconazole
(1 g) was added to each disk. The plates were incubated at 30°C for 24 h and photographed. Scale bar ⫽ 6 mm. (C) Time-killing curve of wild
type and cna1/cna1, cnb1/cnb1, and crz1/crz1 mutants in YPD medium ⫾ ﬂuconazole. The data are represented as means ⫾ SDs from triplicate
810 CHEN ET AL. E
Calcineurin and Crz1 are required for hyphal growth in C.
dubliniensis. It is unclear if calcineurin is required for hyphal
growth in C. albicans. Two groups, including our own, found no
clear role for calcineurin in hyphal growth (5, 8), while another
group suggested that calcineurin and Crz1 may be required for
hyphal growth on spider medium (carbon source starvation)
(42, 68). Sanglard et al. reported that a C. albicans cna1/cna1
mutant also exhibits attenuated hyphal growth on SLAD me-
dium (nitrogen source starvation) (68). Here, we clarify the
roles of calcineurin in C. dubliniensis hyphal growth in re-
sponse to carbon or nitrogen limitation. We found that hyphal
growth of cna1/cna1 and cnb1/cnb1 mutants and FK506-
treated wild type is severely impaired on solid spider, SLAD,
and FA (ﬁlament agar; no added nitrogen source) media (Fig.
6), showing a clearly demonstrable role for calcineurin in hy-
phal growth in response to nutrient deprivation in C. dublini-
ensis. Interestingly, Crz1 is required for hyphal growth during
carbon (spider medium) but not nitrogen (SLAD and FA me-
dia) starvation in C. dubliniensis (Fig. 6). The hyphal growth
defects of C. dubliniensis crz1/crz1 mutants on solid spider
medium were complemented by introducing the wild-type
CRZ1 gene under the control of its native promoter (see Fig.
S2 in the supplemental material). In C. albicans, Karababa et
al. also reported that crz1/crz1 mutants exhibit hyphal growth
defects on solid spider medium, but the roles of C. albicans
Crz1 in growth on nitrogen-limited medium have not been
reported (42, 69). On solid serum agar (50% serum, 2% agar),
C. dubliniensis calcineurin is integral for growth while Crz1 is
not. However, the hyphal growth of crz1/crz1 mutants was
attenuated on serum agar compared with the wild-type or
complemented strains (Fig. 6 and data not shown), suggesting
a speciﬁc role of Crz1 in regulating hyphal growth in solid
We also found that C. dubliniensis cna1/cna1 and cnb1/cnb1
mutants and FK506-treated wild type but not crz1/crz1 mutants
exhibit reduced germ tube formation in liquid spider medium
compared with the wild type (P ⬍ 0.01) (see Fig. S6 in the
supplemental material). In contrast, a C. albicans cna1/cna1
mutant or FK506-treated wild-type cells exhibit normal germ
tube formation in liquid spider medium (Fig. S6), suggesting
that calcineurin function in response to nutrient starvation in
liquid may be diverged between the two species. O’Connor et
al. recently reported that unlike C. albicans, C. dubliniensis
exhibits differential hyphal growth in response to nutrient star-
vation (57). Interestingly, C. dubliniensis Crz1 shows differen-
tial responses to hyphal growth in solid and liquid spider media
(Fig. 6; see also Fig. S6). In contrast to defects on solid spider
medium, C. dubliniensis crz1/crz1 mutants exhibit normal germ
FIG. 4. Calcineurin and Crz1 control cation homeostasis in C. dubliniensis. (A) Cells were grown overnight in YPD at 30°C, 5-fold serially
diluted, and spotted onto YPD medium containing CaCl
, or NaCl at the concentration indicated. The plates were incubated at 30°C for
48 h. (B) Quantitative real-time PCR was used to assay expression of genes involved in cation homeostasis (PMC1 and PMR1) or calcium channel
(CCH1 and MID1) in the wild-type and mutant strains. The fold changes in transcription of each gene were normalized to the endogenous control
ACT1. The error bars represent means ⫾ SDs from a triplicate experiment. One representative ﬁgure of three independent experiments is shown.
Asterisks indicate P ⬍ 0.001 compared with the wild type.
OL. 10, 2011 ROLES OF CALCINEURIN PATHWAY IN C. DUBLINIENSIS 811
tube formation in liquid spider medium compared with wild
type (Fig. S6). These lines of evidence show that calcineurin
(Cna1 and Cnb1) is required for hyphal growth in response to
nutrient starvation, while Crz1 plays different roles dependent
upon nutrient starvation and other medium conditions (solid
or liquid) in C. dubliniensis. These results suggest that the role
of calcineurin in hyphal growth of C. dubliniensis is, at least in
part, mediated by Crz1 in response to various environmental
Thigmotropism is attenuated in the crz1/crz1 mutant. The
thigmotropic (contact-sensing) response of fungal hyphae en-
ables the growing tip to circumnavigate impenetrable objects
encountered in the environment. In plant pathogens, sensing
substrate contours allows fungi to locate and identify the spe-
ciﬁc topography of penetration sites in the host leaf (1). Con-
tact with an object is proposed to be sensed in C. albicans by a
mechanism involving activation of plasma membrane calcium
channels, which initiates a turning response in the hyphal tip
(11, 88). We tested whether calcium signaling through the two
calcineurin subunits or the Crz1 transcription factor was re-
quired for thigmotropism in C. dubliniensis. Because the hy-
phae of C. albicans and C. dubliniensis are similar in diameter,
we tested the response using the same quartz slides with
0.79-m ridges. No difference was observed in the thigmo-
tropic response of the cna1/cna1 or the cnb1/cnb1 mutants
compared to the wild-type strain, but turning was reduced by
approximately one-third in the crz1/crz1 mutant (P ⫽ 0.032)
(Fig. 7). This result is similar to that observed for C. albicans,
although the effect of the crz1 mutation compared to the con-
trol strain was less marked in C. dubliniensis.
The hyphae of ﬁlamentous fungi generally grow toward the
cathode in an applied electric ﬁeld (galvanotropism) (25). In C.
albicans, deletion of CNA1, CNB1,orCRZ1 resulted in the
attenuation of the galvanotropic response (11). However, we
were unable to test the response of C. dubliniensis using this
assay due to its failure to generate hyphae on application of an
electric ﬁeld of 10 V/cm
, although the cells remained viable
in a ﬁeld applied for 6 h and grew as normal in the medium
used when no ﬁeld was present. One possibility is that the ﬁeld
generated an electrolytic product that inhibited growth in C.
dubliniensis but was not fungicidal.
Deletion of calcineurin and Crz1 attenuates virulence in
mice. C. dubliniensis is generally considered to be a less patho-
genic species compared to C. albicans (77). However, it has
also been reported that C. dubliniensis can be more virulent
than C. albicans in a murine systemic infection model (35, 85).
Here, we used C. albicans as the control group to compare its
virulence to C. dubliniensis and determined the virulence of
calcineurin and crz1/crz1 mutants of C. dubliniensis in a murine
systemic infection model. The median animal survival follow-
ing tail vein infection with 5 ⫻ 10
cells is 20 days for C.
dubliniensis and 2 days for C. albicans, respectively, showing a
dramatic virulence difference (P ⬍ 0.0001) between these two
Candida species. In C. albicans, cna1/cna1 and cnb1/cnb1 mu-
tants exhibit strongly attenuated virulence (4, 5, 8, 68), while
crz1/crz1 mutants have either full (62) or slightly reduced (42)
virulence in a murine systemic infection model. Here we
showed that C. dubliniensis cna1/cna1 (YC40 and YC94) or
cnb1/cnb1 (YC87 and YC96) mutants exhibit strongly attenu-
ated virulence compared with the wild type (P ⬍ 0.0001) (Fig.
8A) while crz1/crz1 mutants (YC107 and YC108) exhibit atten-
uated virulence compared with the wild type (P ⬍ 0.002).
However, there is no statistically signiﬁcant difference between
calcineurin and crz1/crz1 mutant-infected mice (P ⬎ 0.3), in-
dicating that both calcineurin and Crz1 affect the virulence of
To determine colonization ability, we performed kidney fun-
gal burden analysis of animals infected with wild type and the
mutants. cna1/cna1 mutants (YC40 and YC94) exhibited 42-
fold-reduced fungal burden in the kidneys compared with the
wild type (P ⬍ 0.01) (Fig. 8B). In contrast, the difference of
fungal burden between cnb1/cnb1 mutants (YC87 and YC96)
and wild type was less pronounced (P ⫽ 0.08) (Fig. 8B). One
cnb1/cnb1 mutant (YC96) exhibited a 44-fold-reduced fungal
burden compared with wild type (P ⫽ 0.02), while another
cnb1/cnb1 mutant (YC87) exhibited a 2.8-fold (lower fold
change is attributable to a single outlier)-reduced fungal bur-
den compared with wild type (P ⫽ 0.2) (Fig. 8B). When the
FIG. 5. Calcineurin is required for serum survival in C. dubliniensis.
(A) Cells were grown overnight in YPD at 30°C, 5-fold serially diluted,
and spotted onto YPD or 50% serum agar plates. The plates were
incubated at 37°C for 48 h. (B) Germ tube formation of wild-type and
mutant strains in the presence of 100% serum. Cultures in the 96-well
polystyrene plates were incubated at 37°C statically for the times in-
dicated. The percentage of cells forming germ tubes was determined
from at least 200 cells. Scale bar ⫽ 40 m.
812 CHEN ET AL. E
outlier animal from the YC87 infection is excluded from the
analysis, the cnb1/cnb1 mutants (YC87 and YC96) exhibited a
42-fold-reduced fungal burden compared with the wild type
(P ⫽ 0.01). The fungal burden of mice infected with crz1/crz1
mutants (YC107 and YC108) was 3.4-fold reduced compared
with the wild type (P ⫽ 0.09) (Fig. 8B). Taken together, mice
infected with C. dubliniensis calcineurin and crz1/crz1 mutants
exhibited a reduced fungal burden overall compared with the
In histopathological analysis, GMS-stained tissues revealed
that the wild type readily forms hyphae and proliferates exten-
sively in tissues around the renal pelvis, while cells of the
cna1/cna1, cnb1/cnb1, and crz1/crz1 mutants were not observed
(Fig. 8C), indicating that hyphal growth may be reduced in vivo
for the calcineurin pathway mutants. In the H&E staining,
tissue damage or necrosis was observed only in animals in-
fected with the wild type and not with the calcineurin or crz1/
crz1 mutants (Fig. 8C).
Calcineurin mutants are unable to establish murine ocular
infection. Candida species were isolated from AIDS patients
with corneal infections (keratitis) (38, 39). Candida keratitis
caused by C. albicans and NACS, including Candida glabrata
and Candida parapsilosis (16, 81), continues to be an important
cause of ocular morbidity, including loss of vision. Although C.
dubliniensis is frequently found in AIDS patients, it is unclear
if C. dubliniensis has the ability to cause keratitis of patients.
FIG. 6. Calcineurin controls colony hyphal growth in C. dubliniensis. Cells were grown overnight and washed twice with ddH
O. Cells were
separated by sonication, counted with a hemocytometer, and then serially diluted to 10
cells/ml. One hundred microliters containing ⬃100 cells
was spread on a variety of ﬁlament-inducing media ⫾ FK506 (1 g/ml) and incubated at 37°C for the number of days indicated. The experiments
were repeated at least three times, and one representative image is shown. Scale bar ⫽ 1 mm.
FIG. 7. The thigmotropic response is attenuated in C. dubliniensis crz1/crz1 but not calcineurin mutants. (A) The thigmotropic response was
determined when the growing tip reoriented against 0.79-m ridges in the substrate. The turning hyphae are indicated by arrowheads. Scale bar ⫽
25 m. One representative ﬁgure from three independent experiments is shown. (B) Bar graph shows the percentage of total hyphae that
reorientated. The error bars represent the means ⫾ SDs.
OL. 10, 2011 ROLES OF CALCINEURIN PATHWAY IN C. DUBLINIENSIS 813
The comparison of virulence between C. albicans and C. dub-
liniensis in murine ocular infection has not yet been reported.
We here investigate the virulence difference between these two
closely related species and test if calcineurin promotes ocular
infection in C. dubliniensis. Mice infected with an inoculum of
cells of C. albicans exhibited visible opacity and surface
opacity in immunocompetent ICR mice (100%, 15/15).
However, C. dubliniensis at an inoculum of 10
did not result
in persistent manifestation of fungal keratitis in immuno-
competent mice, suggesting that C. dubliniensis is less viru-
lent in murine ocular infection. An immunocompromised
mouse model for fungal keratitis is well established for C.
albicans (60, 89) but not yet tested for C. dubliniensis. We thus
administered cyclophosphamide (180 to 220 mg/kg body
weight), a potent inhibitor of lymphocyte proliferation, to mice
on days 5, 3, and 1 prior to inoculation. All corneas (100%,
15/15) infected with 10
cells of C. albicans SC5314 developed
fungal keratitis compared to 26.7% (4/15) of those exposed to
C. dubliniensis CD36 (Fig. 9A). At all time points, keratitis
caused by C. albicans SC5314 was more severe than that
caused by C. dubliniensis CD36 (Fig. 9B). The disease score of
C. albicans keratitis is persistent, while C. dubliniensis keratitis
has a peak at day 3 but drops subsequently, suggesting that C.
dubliniensis is not a successful pathogen compared to its closely
related species C. albicans. This is the ﬁrst demonstration that
compares these two species using a fungal keratitis model.
Corneas infected with C. dubliniensis CD36 wild type
(26.7%, 4/15) and crz1/crz1 mutant (22.2%, 4/18), but not cal-
cineurin mutants (cna1/cna1 and cnb1/cnb1), showed visible
keratitis (Fig. 9), suggesting that C. dubliniensis calcineurin is
required for establishing murine ocular infection. The mean
keratitis score for the CD36 wild-type strain was similar to the
crz1/crz1 mutant. Keratitis caused by CD36 was moderate
grade by day 1 (6.25 ⫾ 0.96) and became more severe by day
3 (8.00 ⫾ 0.82, P ⫽ 0.033) with inﬂammation starting to resolve
by day 4 (5.75 ⫾ 1.26) (Fig. 9B). Fungal keratitis resulting from
the crz1/crz1 mutant started to resolve by day 3 and was sig-
niﬁcantly different by day 6 (P ⫽ 0.027) compared with the
CD36 strain at day 6 (Fig. 9B), suggesting a difference between
CD36 and crz1/crz1 mutant in developing keratitis at later
stages. Compared with the CD36 wild-type and crz1/crz1 mu-
tant strains, it is clear that C. dubliniensis calcineurin mutants
are unable to establish murine ocular infection (Fig. 9), sup-
porting our previous ﬁnding that C. albicans calcineurin mu-
tants exhibit attenuated virulence in a fungal keratitis model
Calcineurin is required for bioﬁlm formation in a rat den-
ture model. In addition to C. albicans and C. glabrata (23), C.
FIG. 8. Calcineurin and crz1/crz1 C. dubliniensis mutants are compromised for virulence in a murine systemic infection model. (A) The survival
of mice following intravenous challenge with 5 ⫻ 10
C. dubliniensis (Cd) or C. albicans (Ca) yeast cells was monitored for up to 42 days. Fifteen
mice per strain were used for all strains except C. albicans wild type (10 mice). (B) The fungal burden in the kidneys was determined at day 7 after
challenge. Five mice per strain were used for all strains except YC107 (4 mice were analyzed because one animal died immediately after infection).
(C) Histopathological sections of kidneys dissected from mice infected with wild-type or cna1/cna1, cnb1/cnb1,orcrz1/crz1 mutant strains. The mice
were challenged with 5 ⫻ 10
yeast cells and sacriﬁced at day 14. GMS and H&E stains were used to observe C. dubliniensis colonization and tissue
necrosis, respectively. Scale bar ⫽ 50 m.
814 CHEN ET AL. E
dubliniensis is frequently isolated from denture wearers who
present with or without denture-related stomatitis (34, 48).
Candida species can form azole-resistant bioﬁlms on dentures,
in which treatment is difﬁcult. However, knowledge regarding
the role of C. dubliniensis in denture bioﬁlm formation is lim-
ited. Therefore, it will be useful to have a denture bioﬁlm
model to examine C. dubliniensis bioﬁlm formation and inves-
tigate calcineurin as a potential drug target. A rat denture
model was chosen for an in vivo bioﬁlm infection model (54).
After 48 h of growth, the C. dubliniensis wild-type (CD36)
strain produced a bioﬁlm spanning a majority of the inoculated
denture surface consisting of yeast, hyphae, and matrix com-
ponents (Fig. 10). In contrast, inoculation of the cna1/cna1
mutant resulted in only a yeast monolayer (Fig. 10), suggesting
that calcineurin is required for hyphal growth in an in vivo
denture bioﬁlm model and could be targeted for therapeutic
Roles of calcineurin and Crz1 in cell wall integrity and drug
tolerance. Here we demonstrate that calcineurin and Crz1
control cell wall integrity and drug tolerance in C. dubliniensis,
suggesting potential merit for calcineurin inhibitors as novel
therapeutic agents. Most antifungal drugs target fungal protein
components on either the cell membrane or cell wall. For
example, azoles inhibit ergosterol biosynthesis in the cell mem-
brane, and echinocandins inhibit ␤-1,3-glucan biosynthesis in
the cell wall (58). In C. albicans and C. glabrata, calcineurin is
required for cell wall integrity (26, 50). The cell membrane or
wall defects caused by calcineurin mutation render antifungal
azoles fungicidal in C. albicans (26). Here we show that cell
membrane/wall integrity is partially mediated by Crz1 in C.
dubliniensis; crz1/crz1 mutants exhibit SDS hypersensitivity in-
termediate between the wild type and calcineurin mutants
(Fig. 1B). However, C. dubliniensis Crz1 does not play a clear
role in response to cell wall perturbation by CFW and Congo
red (Fig. 1B). These lines of evidence suggest that calcineurin
plays an important role governing cell wall integrity which
might involve other cell wall integrity pathways such as the
protein kinase C (PKC) and high-osmolarity glycerol (HOG)
In C. albicans, cell membrane perturbation by ﬂuconazole
can enhance uptake and toxicity of calcineurin inhibitors (26).
Conversely, it is possible that cell membrane defects caused by
calcineurin mutation result in increased azole uptake and
toxicity, leading to synergistic fungicidal activity. Roles for
calcineurin and Crz1 in azole (26, 68) or echinocandin (70)
tolerance have been studied in C. albicans. However, data
showing interactions between calcineurin and other signaling
pathways to regulate drug tolerance are limited. Singh et al.
demonstrated that heat shock protein 90 (Hsp90) physically
interacts with calcineurin and governs echinocandin resistance
in C. albicans, and drug inhibitors of Hsp90 or calcineurin
exhibit synergistic fungicidal activity with echinocandins (at a
nonfungicidal concentration) (70). Recently, LaFayette et al.
showed that PKC signaling regulates azole and echinocandin
tolerance via circuits comprised of calcineurin, Hsp90, and
Mkc1 in C. albicans (46). It is possible that C. dubliniensis
shares these conserved pathways that may function in coordi-
nation with the calcineurin pathway to effect cell wall integrity
and drug tolerance.
Roles of calcineurin and Crz1 in hyphal growth and contact
response orientation. In C. albicans, the roles of calcineurin in
hyphal growth are unclear; two groups, including our own,
were unable to ﬁnd a role for calcineurin in hyphal growth (5,
26), while another group reported that a calcineurin mutant
(cna1/cna1) exhibited hyphal growth defects on spider and
SLAD solid media (68) (Table 3). The contrasting results re-
garding the roles of the calcineurin pathway in hyphal growth
of C. albicans may be due to different C. albicans backgrounds
or experimental details. In this study, we aimed to use C.
dubliniensis, a species closely related to C. albicans, to investi-
gate the roles of calcineurin (Cna1 and Cnb1) in hyphal
growth, a phenotype linked to virulence. We ﬁnd that calcineu-
FIG. 9. C. dubliniensis calcineurin mutants are unable to establish
murine ocular infection. (A) Clinical photographs of corneas of im-
munosuppressed (cyclophosphamide-treated) mice 2 days after the
inoculation of 10
yeast cells. Fungal keratitis, indicated by red arrows,
was seen only in animals infected with C. albicans SC5314 (100%,
15/15), C. dubliniensis CD36 (26.7%, 4/15), and crz1/crz1 mutant
(YC108, 22.2%, 4/18). (B) Each cornea of an immunosuppressed
mouse was inoculated with 10
yeast cells of each strain, and the
disease severity was scored for 8 days. C. albicans SC5314 served as a
reference control. Mice infected with C. dubliniensis cna1/cna1 or
cnb1/cnb1 mutants or the PBS control exhibited normal cornea, and
score curves essentially overlapped. Mice infected with C. albicans
SC5314 and C. dubliniensis wild-type CD36 and crz1/crz1 mutant
strains, exhibiting visible signs of keratitis, were plotted.
OL. 10, 2011 ROLES OF CALCINEURIN PATHWAY IN C. DUBLINIENSIS 815
rin (Cna1 or Cnb1) is clearly required for hyphal growth in
response to either carbon or nitrogen source limitation in C.
dubliniensis (Fig. 6; Table 3). However, in C. albicans we are
unable to appreciate a clear role for calcineurin in hyphal
growth upon either carbon or nitrogen source starvation.
In C. albicans, the roles of the transcription factor Crz1 in
hyphal growth remain elusive. Karababa et al. reported that
Crz1 is required for hyphal growth on spider medium (42),
while Noble et al. showed that crz1/crz1 mutants exhibited no
hyphal growth defects on spider medium from a systematic
FIG. 10. Scanning electron microscopy (SEM) images of a C. dubliniensis rat denture bioﬁlm model. Rat dentures were harvested after 48 h
of growth, processed for SEM, and imaged. Scale bars for 50⫻ and 2,000⫻ images represent 500 M and 10 M, respectively.
TABLE 3. Summary of C. albicans and C. dubliniensis calcineurin and crz1/crz1 mutant phenotypes
C. albicans C. dubliniensis
Calcineurin crz1/crz1 Calcineurin crz1/crz1
Cell wall integrity
SDS Inviable Intermediate
CFW Sensitive Wild type Hypersensitive Wild type
Congo red Wild type Wild type Hypersensitive Wild type
Echinocandin Hypersusceptible Differential
Azole Hypersusceptible Intermediate Hypersusceptible Intermediate
Hypersensitive Hypersensitive Hypersensitive Hypersensitive
Wild type Hypersensitive
Serum survival Hypersensitive Wild type Sensitive Wild type
Hyphal growth (solid surface)
Carbon limitation Wild type Wild type Impaired Impaired
Nitrogen limitation Wild type Wild type Impaired Wild type
Thigmotropism Wild type Attenuated Wild type Attenuated
Galvanotropism Attenuated Attenuated NA
Virulence (murine systemic infection) Attenuated Wild type/attenuated Attenuated Attenuated
Intermediate phenotype between wild type and calcineurin mutants.
Mutants exhibit either no response or intermediate susceptibility to echinocandins.
These observations were found at 24 and 30°C (0.4 M). Interestingly, C. albicans mutants exhibit the wild-type phenotype while C. dubliniensis mutants exhibit the
sensitive phenotype at 37°C (see Fig. S5 in the supplemental material).
Hypersensitivity at 2 M NaCl.
Hypersensitivity at 1 M NaCl.
Failure to generate hyphae when an electric ﬁeld was applied.
816 CHEN ET AL. EUKARYOT.CELL
screen (55). The confounding results may be due to different
experimental methods or genetic backgrounds of C. albicans
strains. However, the calcineurin target CrzA in A. fumigatus
has been demonstrated to regulate hyphal growth (24), indic-
ative of a potential global role of the calcineurin target Crz1/
CrzA in regulating hyphal growth in fungal pathogens. In sup-
port of the interpretation that Crz1 is a global regulator of
hyphal growth, we ﬁnd that Crz1 is required for hyphal growth
on solid spider and serum media in C. dubliniensis (Fig. 6).
However, C. dubliniensis Crz1 is not required for germ tube
formation in either liquid spider or serum medium (Fig. 5; see
also Fig. S6 in the supplemental material), indicating a fasci-
nating role for Crz1 in adhering to a solid surface.
Brand et al. demonstrated that hyphal orientation (thigmot-
ropism or galvanotropism) is linked to the calcium signaling
and calcineurin pathway in C. albicans (9–11). We observed
that thigmotropism was reduced in C. dubliniensis (CD36) by
25 to 30% compared with C. albicans (SC5314) (data not
shown). The defective thigmotropism in the C. dubliniensis
crz1/crz1 mutant but not calcineurin mutants is consistent with
the ﬁndings that thigmotropism is mediated by Crz1 in C.
albicans (11) (Table 3). In C. albicans, loss of the thigmotropic
response correlated with reduced tissue penetration and dam-
age of oral epithelial cells in an in vitro assay (12). Thus, the
attenuated thigmotropism of C. dubliniensis crz1/crz1 mutants
may partly explain their attenuated virulence in a murine
systemic infection model (Fig. 7), but thigmotropism does
not appear to contribute to the attenuated virulence of the
C. albicans and C. dubliniensis calcineurin mutants, in which
attenuated virulence may simply be due to the essential role of
calcineurin for survival in serum in both species.
Role of calcineurin and Crz1 in serum survival and viru-
lence. Fungal pathogens require calcineurin for virulence, but
the precise role of calcineurin is species dependent (18). The
roles of calcineurin in serum survival have been demonstrated
in the human fungal pathogens C. albicans (7) and A. fumigatus
(73). In contrast, in C. neoformans calcineurin supports
growth at mammalian body temperature (37°C) (59). The
plant fungal pathogens M. oryzae (21) and Ustilago maydis
(29) have adapted calcineurin for different pathogenic mech-
anisms involving appressorial formation and ﬁlamentous
growth, respectively (18). Here we demonstrate that calcineu-
rin is required for serum survival in C. dubliniensis (Fig. 5) and,
as a consequence, calcineurin mutants (cna1/cna1 and cnb1/
cnb1) exhibit attenuated virulence (Fig. 8). The requirement
for calcineurin in hyphal growth and cell wall integrity suggests
additional mechanisms by which calcineurin promotes success-
ful infection. Strikingly, C. dubliniensis Crz1 is required for
hyphal growth (Fig. 6) and virulence in a murine systemic
infection model but is not required for serum survival (Fig. 5),
suggesting that Crz1 and calcineurin may contribute to viru-
lence by both common and distinct pathways. In accord with
our observations is the fact that defects in cell wall integrity of
C. albicans often result in attenuated virulence in murine sys-
temic infection models (19, 46, 51).
Our studies also demonstrate that, similar to C. albicans and
Saccharomyces cerevisiae, C. dubliniensis calcineurin and Crz1
are not required for growth at high temperature (see Fig. S3 in
the supplemental material). Thus, calcineurin in C. dubliniensis
does not control virulence through promoting high-tempera-
ture growth, in contrast to the basidiomycete C. neoformans,in
which calcineurin is essential for survival at host body temper-
ature (59). In addition to temperature sensitivity, a Schizosac-
charomyces pombe calcineurin mutant (ppb1) exhibits a cold-
sensitive phenotype associated with cytokinesis defects (91). It
remains largely unknown how calcineurin controls responses to
thermal stress in model or pathogenic fungi.
C. dubliniensis has been isolated from nonhuman sources
such as seabird-associated excrement or ticks, suggesting that
the wax moth (G. mellonella) might be a candidate virulence
model for C. dubliniensis. Interestingly, we found that C. dub-
liniensis is as virulent as C. albicans in the G. mellonella insect
model (P ⫽ 0.32; see Fig. S7 in the supplemental material), in
contrast to their marked virulence difference in the murine
model. All wax moth larvae were dead by day 3, when injected
C. dubliniensis (CD36) yeast cells (P ⬍ 0.0001, com
pared with PBS curve) (Fig. S7), indicating that C. dubliniensis
might be an insect pathogen. However, the roles of calcineurin
and Crz1 in virulence in this insect model do not completely
phenocopy the murine model (data not shown), suggesting that
a speciﬁc niche might be required for the C. dubliniensis cal-
cineurin pathway to be operative during successful infection.
C. dubliniensis is frequently found in the oral cavities of
HIV/AIDS patients; however, its role in this speciﬁc niche is
unclear. To test if C. dubliniensis (CD36) grows and causes
damage to oral epithelium, we used FaDu oral epithelial cells
to analyze cell-host interactions. We found that C. dubliniensis
exhibits less extensive hyphal growth compared with C. albi-
cans (see Fig. S8A in the supplemental material). Spiering et
al. showed that C. dubliniensis grew as yeast for the duration of
the experiment (12 h) in infected reconstituted human oral
epithelium (RHE) (71). We used a
Cr release assay to de
termine if C. dubliniensis causes cell damage. We found that C.
dubliniensis causes no damage while C. albicans triggers dam-
age within 6 h (Fig. S8B). This suggests that C. dubliniensis
might have lost virulence determinants that are necessary to
colonize oral epithelial cells from C. albicans.
Although there are no published clinical reports of keratitis
caused by C. dubliniensis, it is possible that C. dubliniensis
could be an emerging and opportunistic pathogen and cause
ocular infection when the host immune system is compro-
mised. Our keratitis data support this possibility because C.
dubliniensis can cause keratitis in an immunocompromised
host model. Our lab has demonstrated that CsA and ﬂucona-
zole exhibit fungicidal activity against C. albicans in a murine
ocular infection model (60), suggesting a potential combina-
tion therapy for keratitis caused by Candida species.
A summary of the phenotypes of the C. dubliniensis and C.
albicans calcineurin and crz1/crz1 mutants is shown in Table 3.
The C. dubliniensis calcineurin pathway exhibits both con-
served and distinct roles compared with C. albicans. Taken
together, the mechanisms linking calcineurin to C. dubliniensis
pathogenesis involve serum survival and hyphal growth,
whereas the virulence impairment of crz1/crz1 mutants may be
attributable to their defect in hyphal growth. However, it is
possible that other factors such as cell wall integrity may also
contribute to calcineurin and Crz1 effects on pathogenicity.
These lines of evidence suggest that calcineurin could be a
potential drug target in the emerging NACS C. dubliniensis.
VOL. 10, 2011 ROLES OF CALCINEURIN PATHWAY IN C. DUBLINIENSIS 817
We thank Lukasz Kozubowski, Cecelia Shertz, and Joanne Kings-
bury for comments on the manuscript. We appreciate members of the
Heitman and Cardenas labs for helpful discussions. We thank Michael
Lorenz for providing the C. dubliniensis strain CD36 (original source,
Bernard Dujon); Derek Sullivan and Gary Moran for advice on C.
dubliniensis studies; William Steinbach for caspofungin, micafungin,
and anidulafungin; Mitchell Mutz for posaconazole and voriconazole;
Dominique Sanglard for C. albicans calcineurin and crz1/crz1 mutants;
Alice Bungay and Marilou Nicolas for assistance with murine ocular
infection studies; and Joachim Morschha¨user for providing the SAT1
ﬂipper cassette for gene disruptions as well as C. albicans strains.
This research was supported by the Duke University Center for
AIDS Research (CFAR grant 2P30 AI064518-06 to Y.-L. Chen), NIH-
funded program P30 AI64518 to Duke University, and NIH/NIAID
R01 grants AI42159 and AI50438 (to J. Heitman). S. G. Filler and
N. V. Solis were supported in part by NIH grant R01DE017088.
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