Pathway analysis of Candida albicans survival and
virulence determinants in a murine infection model
Jeffrey M. Beckera, Sarah J. Kauffmana, Melinda Hausera, Liyin Huanga, Molly Linb, Susan Sillaotsb, Bo Jiangc,
Deming Xub, and Terry Roemerb,1
a
Department of Microbiology, University of Tennessee, Knoxville, TN 37996; bDepartment of Infectious Disease, Merck Frosst Canada Ltd., Montreal,
QC, Canada H9H 3L1; and cGlycoFi Inc, Lebanon, NH 03766
Edited by Jasper Rine, University of California, Berkeley, CA, and approved October 26, 2010 (received for review July 8, 2010)
One potentially rich source of possible targets for antifungal ther-
apy are those Candida albicans genes deemed essential for growth
under the standard culture (i.e., in vitro) conditions; however, these
genes are largely unexplored as drug targets because essential
genes are not experimentally amenable to conventional gene de-
letion and virulence studies. Using tetracycline-regulatable promo-
ter-based conditional mutants, we investigated a murine model of
candidiasis in which repressing essential genes in the host was
achieved. By adding doxycycline to the drinking water starting
3 days prior to (dox 3D) or 2 days post (dox 2D) infection, the
phenotypic consequences of temporal gene inactivation were as-
sessed by monitoring animal survival and fungal burden in prophy-
laxis and acute infection settings. Of 177 selected conditional shut-
off strains tested, the virulence of 102 was blocked under both
repressing conditions, suggesting that the corresponding genes
are essential for growth and survival in a murine host across early
and established infection periods. Among these genes were those
previously identified as antifungal drug targets (i.e., FKS1, ERG1,
and ERG11), verifying that this methodology can be used to
validate potential new targets. We also identify genes either con-
ditionally essential or dispensable for in vitro growth but required
for survival and virulence, including those in late stage ergosterol
synthesis, or early steps in fatty acid or riboflavin biosynthesis.
This study evaluates the role of essential genes with respect to
pathogen virulence in a large-scale, systems biology context, and
provides a general method for gene target validation and for
uncovering unexpected antimicrobial targets.
animal model antifungal target conditional expression
systemic candidiasis virulence factor
C andida albicans exists naturally as a commensal microorgan-
ism of the human gastrointestinal tract. Subtle alterations
in its relationship with the host can, however, trigger a transition
to a pathogenic state. Indeed, C. albicans represents the most
medically significant human fungal pathogen, causing a variety of
skin and soft tissue infections in healthy individuals and more
virulent invasive and disseminated diseases in a hospital setting,
particularly among patients with compromised immune systems.
Accordingly, its pathogenesis and the resulting spectrum of dis-
ease states are questions of significant biological interest as well
as being of major clinical and economic importance.
Genetic strategies have been used to study the ecology, survi-
val, and virulence determinants of C. albicans. However, because
C. albicans is an obligate diploid and lacks a complete sexual cycle
based on meiosis, it is largely refractory to the classical genetic
analysis used in the bakers yeast, Saccharomyces cerevisiae (1).
Consequently, a number of molecular genetic approaches have
been developed in C. albicans to introduce targeted gene muta-
tions, including the URA Blaster (2), SAT1 flipper (3), and other
cassette methods (4) to construct viable homozygote deletions.
Transposon heterozygosity (5) and regulatable antisense interfer-
ence approaches to modulate gene expression (6) have also been
described. However, these approaches are largely biased toward
constructing nonlethal mutations; deletion analyses of essential
2204422049 PNAS December 21, 2010 vol. 107 no. 51
genes (with the exception of conditional essential genes) and
characterization of their resulting terminal phenotypes both in
vitro and in the host milieu have been largely ignored despite
their central role in cell growth and division of the pathogen.
Moreover, for many gene products, their functional role in cen-
tral cellular processes can depend largely upon the specific envir-
onment(s) in which their phenotypes are assessed. Consequently,
significant differences in growth phenotypes and virulence can
occur between in vitro and in vivo conditions among deletion
mutants, as recently demonstrated among pathogenic Gram-
positive bacteria disrupted for fatty acid synthesis (7, 8).
Microbial pathogens face a barrage of diverse environmental
stresses during colonization of different niches within the host.
These include variable pH, carbon sources, temperature, osmo-
larity, adherence, and other physiological stresses, including the
host's armamentarium of immune responses (9). Accordingly,
extrapolating phenotypes derived from a particular in vitro envir-
onment to those in a host infection setting can be misleading, as
elegantly exemplified by different requirements for growth in
vitro and for virulence in a mouse model among deletion mutants
constructed in a clinically isolated strain of S. cerervisiae (10, 11).
Extrapolating gene function across fungal species should also
be viewed with caution, as remarkable rewiring in regulatory
networks governing carbohydrate utilization, amino acid bio-
synthesis, and ribosomal biogenesis has been demonstrated
across ascomycete species (see ref. 12 and references therein).
Previously, we have reported the utilization of a tetracycline
(Tet) repressible promoter replacement system in C. albicans for
large-scale phenotypic analysis of genes required for growth under
the laboratory conditions (13). Here, we present the characteriza-
tion of 177 selected Tet conditional shut-off strains to compare
their in vitro growth phenotypes versus phenotypic consequences
on growth, survival, and virulence in an immunocompetent
murine model of systemic candidiasis. The in vivo repressing con-
ditions were established prior to, or following, an acute infection
and were maintained for 3 weeks. Accordingly, this strategy was
used to model drug targets suitable for prophylactic or therapeutic
intervention, respectively. In addition to animal survival, clear-
ance of the pathogen in each treatment regiment was quantified
by fungal burden in the kidneys of infected animals. Cellular
processes required for growth in vitro as well as survival and per-
sistence in the host (e.g., RNA processing, translation, secretion,
mitochondrial function) may be differentiated from those essen-
tial for growth in an in vitro environment without attenuating
Author contributions: J.M.B., S.J.K., M.H., B.J., D.X., and T.R. designed research; S.J.K.,
M.H., L.H., M.L., S.S., and D.X. performed research; J.M.B., S.J.K., M.H., L.H., and B.J.
contributed new reagents/analytic tools; J.M.B., S.J.K., M.H., L.H., M.L., S.S., B.J., D.X., and
T.R. analyzed data; and J.M.B., D.X., and T.R. wrote the paper.
Conflict of interest statement: Some of the authors are employees of Merck & Co., Inc., as
stated in the affiliations, and potentially own stock and/or holdings in the company.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: terry_roemer@merck.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1009845107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1009845107
virulence (e.g., cell wall modifying enzymes). A clear distinction
in the essential role of early ergosterol biosynthetic events and
dispensability of later steps for in vitro growth and virulence is
also observed. However, some late stage genes (e.g., ERG2, and
ERG6) are required for virulence but not in vitro growth. Diverse
aspects of cell metabolism, which if impaired causes an auxo-
trophic in vitro growth phenotype, were similarly assessed to dif-
ferentiate pathways suppressed both in vitro and in the host milieu
(e.g., amino acid and biotin biosynthesis) or essential for virulence
(e.g., fatty acid and riboflavin synthesis). We also discuss the sig-
nificance of this work as it relates to antifungal drug target iden-
tification and genetic validation.
Results
In Vivo Experimental Design and Validation. To survey the relation-
ship between C. albicans gene essentiality under standard labora-
tory conditions (i.e., an in vitro environment) versus survival and
pathogenesis in a host (i.e., an in vivo environment), we first
examined survival and virulence phenotypes of Tet-regulatable
conditional mutants in two essential genes, FKS1 and ERG11.
The FKS1 and ERG11 gene products are targeted by echinocan-
din and azole class therapeutics, respectively (14), and serve as
clinically relevant controls whose genetic inactivation should mi-
mic the efficacy of these agents. As expected, under the standard
laboratory conditions, both strains failed to grow in media con-
taining tetracycline (Fig. 1B). In vivo phenotypic consequences
resulting from transcriptional repression of these genes were
then evaluated by infecting mice with 1 106 cells of either
pTET-FKS1 or pTET-ERG11 in an immunocompetent mouse
model of candidiasis (see Materials and Methods). Three groups of
mice (5 in each) were infected with these strains (Fig. 1C, D). In
the first group (sugar), animals were fed with water supplemented
with 5% sucrose, and all mice succumbed to lethal infection; i.e.,
none survived on day 21 post infection (p.i.) (S21;sugar
0%) and
60% of the animals died within one week (i.e., t60% 7  1 day)
(Fig. 1 C and D). These results were similar to those obtained
with the parental C. albicans strain (Fig. 1E) and other mutants
(Fig. S1), and demonstrated that the pathogenicity and virulence
of these strains is not compromised by promoter replacement in
FKS1 and ERG11 under the nonrepressing conditions. Impor-
tantly, doxycycline (an analogue of tetracycline with superior
pharmacokinetic properties) did not affect virulence of the par-
ental wild-type strain when administered in the drinking water
of infected animals (Fig. 1E). In the second group (dox 3D),
animals were similarly fed with water supplemented with
2 mgmL doxycycline on day 3 prior to infection and thereafter
until day 21 p.i. Doxycycline was also added to the drinking water
of animals in the third group (dox 2D) two days post infection,
and thereafter until day 21. Under the doxycycline-repressing con-
ditions, neither the pTET-ERG11 nor pTET-FKS1 strain led to a
lethal infection in the dox 3D group; i.e., S21;dosx3D
100%,
in two independent sets of experiments, whereas 100% or 80%
survival rates were reproducibly observed in the dox 2D group
for both strains (Fig. 1D, p < 0.001 in all cases, compared with
the corresponding sugar groups). No gross physiological or
behavioral abnormality was observed among surviving animals.
These results recapitulate the essentiality of ERG11 and FKS1
in a systemic candidiasis infection, demonstrating the ability to
reverse an acute disease state by genetic inactivation of these
virulence determinants and in so doing, mimic the effects of their
target-specific antifungal agent.
To quantify pathogen survival and/or clearance by the host in
cases where mice survived infection in either dox 3D or 2D
model, C. albicans colony forming units (CFU) were enumerated

A ON B pTET-ERG11 pTET-FKS1
C
100
pTET ERG11
p YPD YNBD RPMI+S YPD YNBD RPMI+S 80 sugar
% survival

HIS3 60 dox-3D
ON
dox+2D

APPLIED BIOLOGICAL
40
OFF dox-3D/W
pTET ERG11 OFF 20 dox+2D/W

SCIENCES
HIS3
Tet 0
0 7 14 21 28 35
days post infection
D 100% survival 0
F
7 14 % D21
sugar 0
dox-3D 100 days post infection 21 35 21 35
dox+2D 80
ERG11 sugar 0 infection model dox-3D dox+2D
dox-3D 100
dox+2D 80
log10(CFU/g kiidney)

sugar 0
dox-3D 100
dox+2D 100 6
FKS1 0
sugar
dox-3D 100
dox+2D 80
4

dox 2
E sugar
pTET-ERG11 pTET-FKS1

Fig. 1. Identification of the essential role of ERG11 and FKS1 in a murine model of candidiasis using conditional shut-off strains. (A) The Tet-off system used to
construct conditional shut-off strains. The active Tet-transactivator is indicated by a filled diamond, inactive open symbol, and tetracycline red dot. (B) The
terminal phenotypes of indicated strains under the repressing conditions (100 gmL tetracycline) on media indicated. S 20% mouse serum. (C) Standard
survival curves of animals infected with the pTET-ERG11 strains under different conditions, sugar, nonrepressing; dox 3D, repressing conditions established
3 days prior to infection; dox 2D, established 2 days p.i.; dox 3DW, nonrepressing conditions from days 2235 for surviving animals from the dox 3D
group; dox 2DW, for animals from the dox 2D group. Arrows indicate necropsy and fungal burden determination on days 21 and 35 p.i. (D) Summary of
independent animal experiments with the conditional shut-off strains of ERG11 and FKS1, with each experiment consisting of three conditions indicated on the
left. The survival rates are represented by red color with different shades, with scale shown on the top. The survival rates on day 21 are on the right. Green bars
indicate the day on which 60% of the animals expired due to lethal infection. (E) Effects of doxycycline on the in vivo virulence of the parental strain used in
this study. Animals were fed with sugar or doxycycline immediately after infection and thereafter. (F) Kidney fungal burdens (days 21 and 35 p.i.) of animals
infected with the pTET-ERG11 and pTET-FKS1 strains under the two repressing conditions, dox 3D and dox 2D. Dashed horizontal line indicates limit of
detection in enumerating fungal burden.

Becker et al. PNAS December 21, 2010 vol. 107 no. 51 22045
from infected kidneys of two sacrificed animals. Whereas wild-
type C. albicans strains normally achieve a kidney burden be-
tween 1 106 107 CFUg within day 7 p.i. (Fig. S2 and refs. 15
and 16), fungal burdens achieved by the pTET-FKS1 and
pTET-ERG11 strains were under the limit of detection
(<103 CFUg) or minimal (104 CFUg) on day 21 p.i. in ani-
mals from the dox 3D group (Fig. 1F). Higher kidney fungal
burdens were observed in the dox 2D group, ranging from
<103 to 5.0 105 CFUg (Fig. 1F). The surviving animals in both
groups were kept for two additional weeks on regular drinking
water and all mice survived throughout this extended (day 35)
time period (Fig. 1C and Table S1Table S3). Two animals from
each group were selected for necropsy. The kidney burdens of the
pTET-FKS1 strain in the dox 3D group remained under the
limit of detection, indicating likely eradication of the fungal in-
fection. Low levels (<105 CFUg) of the pTET-ERG11 strain
were observed under the same repression conditions (Fig. 1F).
Fungal burden levels of both strains were notably higher
(>1.0 106 CFUg) in all but one of the dox 2D animals at
day 35, reflecting a marked (but incomplete) fungal eradication
during doxycycline treatment in the dox 2D setting but poten-
tial recolonization of the host subsequent to the 2 week duration
without repressor (Fig. 1F). Collectively, these results confirm
that regulatable repression offered by the Tet (Dox)-regulatable
conditional mutant strains is achieved and can be extended in vivo
to a systemic infection model, enabling in vitro versus in vivo
phenotypic relationships to be directly assessed. Moreover, tem-
poral regulation of C. albicans virulence determinants in an in
vivo setting provides important insights into their phenotypic
consequences in initiating and/or maintaining an established in-
fection as well as their requirement for pathogen survival and/or
clearance from the host.
Surveying C. albicans Survival and Virulence Determinants. Applying
this infection model, we performed a large-scale screen of
Tet-based conditional promoter mutants to identify C. albicans
virulence determinants required for in vivo survival across 175
additional genes predicted to function in various aspects of cell
growth, division and metabolism (Table S1). Genes were selected
according to their growth phenotype under in vitro conditions, as
well as their conservation in Aspergillus fumigatus [a filamentous
fungus of significant medical importance (17)] and absence or
minimal conservation in humans. Based on the ERG11 and FKS1
data (Fig. 1), we deemed genes essential in vivo if the correspond-
ing strains were virulent in the sugar group but failed to cause
lethal infection in 80% animals in both the dox 3D and dox
2D groups in 3 weeks (i.e., S21;dox3D
80% and S21;dox2D

80%). A total of 102 essential genes (including ERG11 and
FKS1) were identified (with p < 0.001 in all cases, compared with
the corresponding virulent control groups), of which 64 corre-
sponding strains were completely avirulent (S21;dox3D
100%
and S21;dox2D
100%) under both repression conditions
(Fig. 2A and Tables S1 and S2). On the other hand, 29 genes
were found to be dispensable to candidiasis in our model, with
S21;dox3D
20% and S21;dox2D
20%. In all cases, no statistical
difference was observed between the two repressing conditions
and the corresponding sugar groups. For the remaining 46
strains, the animal survival rates were <80% but 40% under
either of the repressing conditions (Fig. 2A and Tables S1 and
S2). For all but one strain (ERG4), the animal survival rates of
the dox 3D groups were statistically higher (p < 0.001) than
the corresponding sugar groups (Table S1 and Fig. S1). With the
exception of 4 strains, the overall survival rates of the dox 3D
groups were significantly greater (p < 0.05) than the correspond-
ing dox 2D groups (Table S1 and Fig. S1). In particular, 24
strains in this class were completely avirulent under the dox 3D
conditions (S21;dox3D
100%; p < 0.001) and their virulence
was significantly attenuated in the dox 2D groups (Fig. 2A
and Table S1). Genes with attenuated virulence are not restricted
to particular functional groups (Fig. S3).
A quantitative difference between essential genes and those
with attenuated virulence is confirmed by the kidney fungal bur-
dens (on day 21 p.i.) of infected animals (Fig. 2B and Table S3).
Under the dox 3D repressing conditions, the fungal burdens
of strains of essential genes were under the limit of detection
in 70% of animals, and 5% animals sustained >106 CFUg
kidney burdens. The opposite was observed with strains of genes
with attenuated virulence when repressed. Approximately 40% of
the animals showed sign of eradication under the same repressing
conditions, whereas more than 30% animals suffered fungal bur-
dens >106 CFUg kidney (Fig. 2B). Furthermore, the delay in
establishing the repressing conditions (i.e., dox 2D) decreased
the portion of animals with <103 CFUg kidney by 50% for
strains in both classes, with a concomitant increase (5-fold) in
animals with significant fungal burdens (>106 CFUg) of strains
of essential genes. The incidence of animals with similar fungal
burdens of strains with attenuated virulence increased as well
from 30% to 50% (Fig. 2B). These quantitative changes were
not manifested in significant changes in S21;dox2D
of animals
infected with strains of essential genes. However, for strains with
attenuated virulence, the S21;dox2D
of great majority (42 out
of 46) was statistically lower (p < 0.05) than the corresponding

A dox+2D (outer) B 80 C
n = 29 attenuated dox-3D CE
26 attenuated dox+2D 100 NE
60
percentage

essential dox-3D = //
26 E
number

3 64 essential dox+2D 40
4 10 40
dox-3D 101
5 11 (inner) 98
4 20 34 19
4 24 20
3
4 5 10
16 0
essential

non-essential
S21 80%

(N=11)
S21 <80%
(N=35)

3
(N=102)

(N=29)

34
34 0
n = 46 n = 102
3.0 4.0 5.0 6.0 7.0 > 7.0
100% survival 0
lg (CFU/g kidney)
attenuated

Fig. 2. Virulence phenotypes of 177 C. albicans genes. A. Graphic summary of animal survival rates. Genes are divided into three classes, essential (S21;dox3D

and S21;dox2D
80%); with virulence attenuated (40% S21;dox3D
and S21;dox2D
< 80%), and nonessential (S21;dox3D
and S21;dox2D
20%). For each class,
the survival rates under the dox 3D conditions are presented proportionally (indicated by numbers of strains) in the inner donut with the corresponding
dox 2D survival presented in the outer donut. (B) Distribution of kidney fungal burdens (CFUg, on day 21 p.i.) of animals infected with strains of essential
genes (red) and those with attenuated virulence (green) under the repressing conditions of dox 3D (open) and dox 2D (filled). Data used in the analysis are
indicated in Table S3. (C) Concordance of in vivo virulence and in vitro growth phenotype. Genes are divided into four classes, and the corresponding in vitro
phenotypes in each class are presented proportionally (indicated by number of strains) by E (essential), NE (nonessential), and CE (conditional-essential, defined
as essential on YNBD but not on YPD). A summary of all animal survival curves and p-values for C. albicans strains corresponding to genes with attenuated
virulence is provided (Fig. S1).

22046 www.pnas.org/cgi/doi/10.1073/pnas.1009845107 Becker et al.

S21;dox3D
(Table S1 and Fig. S1) We examined the fungal bur-
dens on days 2, 4 and 7 in the dox 2D model of infection, and
determined that statistical differences in fungal burdens between
animals infected with strains for essential genes and those with
attenuated virulence were observed only after the repressing con-
ditions were established (Fig. S2), suggesting that the different
infectivity of these two groups of strains is not due to the initial
burdens. These data demonstrate the quantitative and dynamic
aspects of in vivo virulence as determined by individual genes
and the repressing conditions.

Concordance of in vitro Growth Phenotype and Virulence. The term-

inal growth phenotypes of all the conditional shut-off strains used
in this study were determined on solid media containing tetracy-
cline at 30 C (Table S2). With few exceptions, growth phenotypes
scored at 30 C were reproduced at the physiological temperature
(37 C) (Fig. S4). We then compared the virulence results with
the corresponding in vitro phenotypes under the repressing con-
ditions and observed a strong correlation (Fig. 2C). With only
one exception (ERG6), all the strains of genes essential for in
vivo virulence failed to grow or displayed severe growth defects
(Table S2) on YNBD agar with tetracycline (Fig. 2C). In contrast,
of the 29 genes not essential for virulence, 10 were also dispen-
sable in vitro, whereas the remaining 19 were all conditionally
essential depending on the in vitro medium used to assay growth
phenotypes; i.e., they conferred no or minimal growth defects un-
der the repressing conditions on YPD (i.e., rich) but are essential
on YNBD (i.e., minimal) (Fig. 2C and Table S1). Genes with
attenuated virulence could be divided into two subgroups, those
with S21;dox3D
80% and those <80%. All but one of 35 genes
in the first subgroup were essential for viability in vitro. In the
second subgroup, 3 were essential in vitro, 5 were not, and 3
was conditional-essential (Fig. 2C and Table S1). The high degree
of concordance (97%, if in vitro essentiality was determined on
both YPD and YNBD, 85% if only YNBD) between the growth
phenotype of in vitro transcriptional repression and the virulence
under in vivo repressing conditions established prior to infection
(dox 3D) is a further indication that the Tet-off system can be
used to efficiently inactivate the expression of genes of interest in

APPLIED BIOLOGICAL
an animal host setting. Corroborating this view, we determined

SCIENCES
that all isolates recovered 4 and 7 days after animal infection
were phenotypically indistinguishable from the starting strains
under the nonrepressing and repressing conditions, indicating
no obvious genetic changes to the conditional promoter system
during infection of in a host setting (Fig. S2).

Pathway Analysis of Virulence Determinants. Whereas virulence fac-

tors are defined as nonessential for growth in vitro but directly
involved in pathogenesis (e.g., toxins, proteases, and signaling
pathways), virulence determinants are essential for growth in a
host setting and may indirectly affect such pathogenesis-causing
processes (18). As expected, our analyses demonstrate a broad
range of gene ontology (GO) functional processes that serve as
virulence determinants, including DNA replication, RNA synth-
esis, translation, mitochondrial function, protein secretion, and
cell wall biosynthesis (full description in SI Text). However, per-
forming such in vitro-in vivo phenotypic analysis systematically
across all steps within a specific pathway yielded unexpected find-
ings, as demonstrated within the ergosterol pathway.
Many of the most clinically relevant antifungal agents (e.g.,
fluconazole, voriconazole, posaconazole) target ergosterol bio-
synthesis, with the genetic and biochemical characterization of
this pathway being one of the most extensively studied aspects
of C. albicans physiology. A molecular characterization of the
C. albicans ergosterol pathway, however, is largely based on
inferences derived from studies in S. cerevisiae, with direct genetic
analyses in the pathogen being restricted to a subset of nonessen-
tial steps in this pathway. Therefore, conditional mutants of all 20
Fig. 3. Summary of survivals of animals infected with the C. albicans condi-
tional shut-off strains of genes involved in the ergosterol pathway. Genes are
listed according to their roles in the pathway (Left). The growth phenotypes
of the corresponding strains on YPD (1% ethanol, the nonrepressing condi-
tions) and YPD TET (100 gmL tetracycline and 1% ethanol, repressing) at
30 C (2 days) are shown in the middle. Survival data are summarized on the
right. Each strain was used to infect animals under three conditions (5 ani-
mals each): S, fed with sugar in the drinking water (nonrepressing); 3D and
2D, fed with doxycycline in the drinking water (repressing) 3 days prior to
and 2 days post infection, respectively. Animal survivals (red with different
shades, with the scale shown on top) were monitored for 3 weeks (separated
by white bars). The statistic significance of survival under the each repressing
condition was compared with that in the corresponding sugar group, ***,
p < 0.001; and **, p < 0.01. Those under two repressing conditions were also
compared, with indicating p < 0.001; p < 0.01; and p < 0.05.
genes participating in ergosterol synthesis were evaluated for in
vitro versus in vivo growth phenotypes (Fig. 3 and Fig. S5). All

Becker et al. PNAS December 21, 2010 vol. 107 no. 51 22047
steps acting early in ergosterol precursor synthesis, spanning
acetyl-CoA thiolase (ERG10), mevalonate synthesis (ERG13,
HMG1), isoprenyl-pyrophosphate synthesis (ERG12, ERG8,
MVD1) and its conversion to squalene (ERG9, ERG20), were
essential for growth in vitro and virulence in both dox 3D and
dox 2D models of infection (p < 0.001). Steps required for the
conversion of squalene to its cyclized sterol, lanosterol (ERG1,
ERG7), and subsequent 14-demethylation (ERG11) were also
essential for growth and virulence in both models (p < 0.001),
consistent with the antifungal effects of terbinafine and azoles,
targeting ERG1 and ERG11, respectively (19). Conversion of the
Erg11p enzyme product (4,4-dimetyl-8,14,24-trienol) to zymoster-
ol requires ERG24ERG27; all of which are essential for S. cere-
visiae growth (20). ERG24, although not essential for growth in
vitro, is required for virulence in both infection models
(p < 0.01) as previously shown (21). We also corroborated pre-
vious findings that ERG27 is essential for growth in vitro (22)
and demonstrate its requirement for survival and virulence in both
infection models (p < 0.001). ERG26, however, was reported to
be essential (23), conflicting with our findings, likely due to an
insufficient repression of the Tet promoter in this instance
(Fig. S6; see Discussion). We also failed to verify the essentiality
of ERG251/orf19.4631 as demonstrated in S. cerevisiae, likely
due to an ERG25 paralog (ERG25/orf19.3732) in C. albicans. In
S. cerevisiae, later biochemical steps involved in the conversion
of zymosterol to ergosterol are not essential for growth. This was
also the case for conditional mutants corresponding to ERG2
ERG6; growth was largely unaffected in vitro under repressing
conditions. Interestingly, conditional mutants of ERG2, ERG3
and ERG6 were significantly less virulent in the dox 3D infection
model (p < 0.001). Further, the pTET-ERG2 and pTET-ERG6
conditional mutants demonstrated similar avirulent phenotypes
under the dox 2D conditions. These results corroborate the
previous finding that ERG3 is required for virulence (24, 25)
and expands this conclusion to additional late stage ergosterol
biosynthetic enzymes even though they are dispensable for growth
under in vitro conditions.
Discussion
We surveyed the effects on virulence and pathogen survival result-
ing from transcriptional repression of 177 distinct C. albicans
genes in a murine infection model of systemic candidiasis. Unlike
deletion mutants, whose genetic inactivation is fixed throughout
the infection process, or alternative conditional mutant strategies
that rely on regulatable promoters that are constitutively repressed
in a host environment (26, 27), Tet-based conditional mutants
offer temporal repression of a query gene at different time points
during infection. Accordingly, administering the repressor to
mice infected with such mutants enables analysis of the phenotypic
consequences of genetic inactivation early in the infection process
as well as later stages of an established C. albicans infection.
Notwithstanding the possible limitations of extrapolating these
data from a mouse model to man, this approach provides a genetic
strategy to predict the potential efficacy of new target-specific anti-
microbials as potential prophylactic or therapeutic agents.
Genes selected for this study participate in diverse cellular
functions and are largely essential for growth of C. albicans under
the in vitro conditions. Perhaps not surprisingly, core housekeep-
ing functions such as mRNA transcription, rRNA and mRNA
processing, ribosomal subunit assembly, and protein secretion
are essential for survival and pathogenesis in a mammalian host.
As the majority of genes evaluated in this study are similarly
essential for growth in S. cerevisiae (20), it is certainly not unex-
pected that C. albicans orthologs often display highly related
terminal phenotypes in vitro and that such genes are also essential
for survival in an animal host. However, inactivation of many
other genes evaluated in this study yield auxotrophic phenotypes
for specific metabolites that are potentially available to the patho-
22048 www.pnas.org/cgi/doi/10.1073/pnas.1009845107
gen in a host environment (see below). Moreover, multiple genes
evaluated in this study are members of gene families (Table S2)
that may be differentially regulated in vivo to buffer the pathogen
from mutant phenotypes otherwise observed in an in vitro setting.
We also identify genes (notably those involved in late stage ergos-
terol biosynthesis such as ERG2 and ERG6), which are dispensa-
ble for viability in vitro but contribute to survival and virulence,
albeit quantitatively. Accordingly, there is no substitute to testing
such mutants in vivo to determine which genes and processes are
important (either directly or indirectly) for virulence and survival
in the host. This work significantly expands the number of viru-
lence-associated genes reported in the literature (n 163; see
Table S4) and broadens their repertoire of functions to include
diverse essential cellular processes.
An important limitation to our study relates to genes deemed
nonessential for pathogen survival and virulence in a host setting.
Although in some instances such genes are dispensable for in vivo
survival (e.g., PHR2; see SI Text and refs. 28 and 29), the failure to
identify a virulence phenotype may reflect an insufficient shut-off
of gene expression by the Tet-promoter system, as demonstrated
by ERG26 (Fig. S6). In principle, this limitation may also extend
to some genes whose repression causes an attenuated virulence
phenotype. Even though such genes are required for virulence in
the dox 3D infection model, we can not discriminate whether
they are required specifically during early events in the infection
process (e.g., successful dissemination within the host or adher-
ence, and subsequent colonization to target organs), or whether
their pathogenicity in the dox 2D group reflects an insuffi-
ciently rapid shut-off of gene expression, high protein half life,
elevated fungal burdens, and/or advanced animal morbidity in
this acute model of infection.
Conditional-essential genes that are potentially rescued by
specific metabolites present in a host environment constitute a
particularly relevant gene set for which in vivo studies are war-
ranted to determine their impact on pathogenesis and potential
suitability as antimicrobial drug targets. Indeed, the therapeutic
efficacy of azoles against Candida glabrata is affected by the
pathogens ability to utilize host cholesterol in the absence of
ergosterol biosynthesis, and neither ERG11 nor ERG9 is essential
in a murine infection model despite both genes being confirmed
essential for in vitro growth of this pathogen (30, 31). Thus
beyond ergosterol (and amino acid biosynthesis), our study in-
cludes 17 additional genes reported as auxotrophic mutants un-
der in vitro growth conditions (either in C. albicans or S. cerevisiae
orthologs; Tables S1 and S2) and rescued by diverse metabolites.
Of these, 13 were demonstrated as essential or attenuated for
virulence in our models. Notable examples include genes re-
quired for early metabolic precursors in cell wall synthesis (GFA1/
glucosamine, GNA1/N-acetylglucosamine), riboflavin (RIB1,
RIB2), and fatty acid biosynthesis (FAS1, FAS2, and OLE1). Ap-
plying such a strategy predicts the achievable efficacy of selective
inhibitors to such targets, as well as mitigates the risk of new anti-
microbial agents lacking efficacy due to target-related reasons.
Bacterial fatty acid biosynthesis provides a salient example of this
concern; despite the development of reportedly efficacious agents
to this pathway, significant controversy remains over whether the
target pathway is essential among Staphylococci for survival and
virulence or whether it is suppressed through scavenging host
fatty acids (7, 8).
Despite significant efforts in the field, few well accepted novel
antifungal targets have been discovered. Essential proteins,
such as Fks1p, that are broadly conserved among relevant fungal
pathogens but absent from humans, constitute the gold standard
spectrum suitable for developing target-selective antifungal
agents. However, other essential proteins, such as Erg11p, that
are common to fungi and man, could also serve as antifungal
targets provided fungal-selective inhibitors can be identified. Con-
sequently, the conservation among all gene products evaluated in
Becker et al.
 200
cans vs. A. fumigatus
150
-lg (BLAST E-value)
essential
100
attenuated
non-essential
ERG11
C. albic
50 FKS1
ERG1
EFT2
0
CDC60
0 50 100 150 200
-lg (BLAST E-value)
C. albicans vs. H. sapiens
Fig. 4. Summary of the 177 C. albicans genes characterized in this study in
the context of their protein sequence similarity to A. fumigatus and human
homologs. The similarity is expressed by lgBLAST E-value; i.e., the higher
this value, the higher the similarity, with 200 including those that are great
than 200. The absence of homolog is indicated by 0. The in vivo virulence
phenotype is represented by different colors; with known targets of antifun-
gal drugs as indicated.
this study were compared to their best Aspergillus fumigatus and
human matches, and their resulting BLAST scores plotted to
reflect the bioinformatic space in which these 177 proteins
reside (Fig. 4). These analyses highlight the rarity of novel proteins
that meet the widely held criteria of a broad spectrum antifungal
target versus the significantly larger pool of functionally character-
ized proteins (e.g., FAS1,2 RIB1,2, ARO1,2,7, and MEX67) that
similarly meet the bioinformatic and phenotypic standards as
FKS1. Imposing a bioinformatics-based fungal selectivity also
markedly restricts this target space, despite their compelling in
vivo target validation by genetic means and resulting clearance
from the host. For example, over 20 genes described in this study
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share human orthologs but produce in vivo terminal phenotypes
reflecting complete clearance of the infection (i.e., fungal burden
below the limit of detection) across dox 3D and dox 2D
treatment regiments at day 21, as well as following a 2 week course
without repressor (Table S3). However, antimicrobials can be
chemically optimized to selectively target pathogen vis--vis ortho-
logous mammalian proteins. For example, azoles, terbinafine
(ERG1) and AN2690 (a novel antifungal agent entering Phase III
clinical trials and targeting the leucyl-tRNA synthetase, CDC60;
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conservation in humans (Fig. 4). Similarly, broad spectrum anti-
bacterials such as antifolates (dihydrofolate reductase), quino-
lones (DNA gyrase and topoisomerase IV), and rifampicin
(RNA Pol II) target proteins with functional orthologs present
in humans. Therefore, screening a broader target space than gen-
erally consideredprovided novel targets are experimentally de-
termined essential for survival and virulenceshould benefit
antimicrobial drug discovery.
Materials and Methods
C. albicans tetracycline-regulatable promoter replacement strains were
constructed using a PCR-based two step conditional mutation strategy as
previously described (13). C. albicans gene annotation and nomenclature
are used according to Braun et al. (33). Strain identification numbers are
provided in Table S2 and are available for noncommercial use to academic re-
searchers following the standard Merck Material Transfer Agreement and
clearance procedures. Detailed assay conditions for the murine systemic infec-
tion model of candidiasis are provided in SI Text, SI Materials and Methods.
ACKNOWLEDGMENTS. We thank previous employees of Mycota Biosciences
and Elitra Canada for their contributions in constructing the C. albicans
strains. We also thank Julian Davies for his thoughtful suggestions and
support throughout the initial stages of this study. Thanks are due to Charlie
Boone, Howard Bussey, Cameron Douglas, and Tim Meredith for their critical
reading of the manuscript, and the two anonymous reviewers for their
constructive comments and suggestions that improved our manuscript. This
work was supported in part by Genome Canada and Genome Quebec.
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PNAS December 21, 2010 vol. 107 no. 51 22049