Self-Regulation of Candida albicans
Population Size during GI Colonization
Sarah Jane White1¤a, Ari Rosenbach1, Paul Lephart1¤b, Diem Nguyen1, Alana Benjamin1¤c, Saul Tzipori2,
Malcolm Whiteway3, Joan Mecsas1, Carol A. Kumamoto1*
1 Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts, United States of America, 2 Division of Infectious Diseases, Tufts University,
Grafton, Massachusetts, United States of America, 3 Genetics Group, Biotechnology Research Institute, National Research Council, Montreal, Quebec, Canada
Interactions between colonizing commensal microorganisms and their hosts play important roles in health and
disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To
gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene,
encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract.
Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of
commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract
and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into
the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other
mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal
colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during
laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine
intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring
commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s)
that regulates gene expression during host colonization can differ from well-characterized pathways used during
Citation: White SJ, Rosenbach A, Lephart P, Nguyen D, Benjamin A, et al. (2007) Self-regulation of Candida albicans population size during GI colonization. PLoS Pathog 3(12):
The opportunistic fungal pathogen Candida albicans colo-
nizes its host long before disease arises. In humans, C. albicans
colonization of the oral cavity is detected in most infants by
the age of one month . The majority of adults are
detectably colonized in the intestinal tract by C. albicans .
Colonization is believed to persist for long periods of time ,
and in this situation, C. albicans is primarily nonpathogenic.
However, if the host becomes immunocompromised, disease
caused by dissemination of commensal organisms from the
intestinal tract can occur. For example, disseminated
candidiasis occurs in neutropenic patients, and Candida spp
are among the most common organisms isolated from the
blood of hospitalized patients . In AIDS patients, orophar-
yngeal candidiasis (OPC) is a common opportunistic in-
fection . Despite the importance of commensal organisms
as the source of infection, little is known about C. albicans
factors that influence intestinal colonization.
A growing body of literature indicates that the interplay
between commensal organisms and the host GI tract is
characterized by reciprocal regulatory interactions. In addi-
tion to a role in nutrition, normal flora are required for
proper development of the intestinal capillary network as
well as Peyer’s patches and other components of the intestinal
immune system . Normal flora stimulate host toll-like
receptors, and these interactions are important for regu-
lation of the inflammatory response. In the absence of toll-
like receptors, dysregulation of the inflammatory response
with concomitant damage to the GI tract occurs . Thus, the
flora make important contributions to the health of the host.
The host also influences its flora. Bulk movement of
material through the GI tract regulates populations of
commensal organisms , and host secretions and immune
effectors play key roles in regulating colonization and
determining the composition of the flora . Therefore,
reciprocal interactions between the commensal flora and the
host maintain the balance between overexuberant inflamma-
tion and uncontrolled growth of microorganisms.
To gain insight into the activities of C. albicans that are
important for host colonization or disease, studies of C.
albicans gene expression during interaction with a host or host
Editor: Brendan P. Cormack, Johns Hopkins University School of Medicine, United
States of America
Received April 16, 2007; Accepted October 22, 2007; Published December 7, 2007
Copyright: ? 2007 White et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: CFU, colony forming unit; CI, competitive index; IGB, immunosup-
pressed gnotobiotic; OPC, oropharyngeal candidiasis; qRT-PCR, quantitative real-
time reverse transcriptase PCR; qPCR, quantitative PCR; WT, wild type; yEGFP, yeast-
enhanced green fluorescent protein
* To whom correspondence should be addressed. E-mail: carol.kumamoto@tufts.
¤a Current address: Department of Chemistry, Massachusetts Institute of
Technology, Cambridge, Massachusetts, United States of America
¤b Current address: Clinical Microbiology/Microbiology and Immunology, Univer-
sity of Rochester School of Medicine and Dentistry, Rochester, New York, United
States of America
¤c Current address: Santa Rosa Family Medicine Program, Santa Rosa, California,
United States of America
PLoS Pathogens | www.plospathogens.orgDecember 2007 | Volume 3 | Issue 12 | e1841866
cells have been pursued. Microarray studies analyzing
interactions between C. albicans and cultured immune cells
have detected dramatic changes in C. albicans metabolism and
stress responses [9–11]. For example, genes encoding enzymes
of the glyoxylate cycle are highly expressed in phagocytosed
fungal cells, and isocitrate lyase is important for systemic
virulence . A study of antigens expressed during oral
infection revealed that components of a MAP kinase signal
transduction pathway were expressed . In addition, the
Not5 protein is expressed during oral infection and is
required for systemic virulence. Recent analysis of C. albicans
cells invading host parenchymal tissue revealed changes in
expression of numerous genes, demonstrating that invading
cells experience metabolic changes and initiate responses to
stresses such as iron limitation .
The goals of this study were to identify C. albicans genes that
were expressed during growth within a host and to compare
the genetic requirements for infection and intestinal colo-
nization. Therefore, genes expressed in C. albicans cells
associated with oral infection or in cells growing in the
intestinal tract were identified, and the ability of mutants
lacking some of the genes to colonize the murine intestinal
tract or to produce disease was characterized. Mutants
lacking the EFH1 gene, encoding a putative transcription
factor , colonized the intestinal tract at higher levels than
wild-type (WT) organisms. Strains engineered to overexpress
EFH1 exhibited reduced levels of intestinal colonization and
reduced colonization of the oral cavity of immunodeficient
(athymic) mice. Therefore, EFH1 is important for determin-
ing the population size of C. albicans during colonization.
Despite its importance during intestinal colonization, EFH1
did not affect virulence in the disseminated infection model
or cellular physiology under laboratory conditions. In
contrast, RBT1, encoding a putative cell wall protein, and
RBT4, encoding a possible secreted protein, are required for
normal virulence in the disseminated infection model 
but are not required for normal intestinal colonization,
despite their high expression during growth in the intestinal
tract. Therefore, genes that influence commensal coloniza-
tion can be distinct from genes that are required for
C. albicans Gene Expression during Growth within an
Immunosuppressed Mammalian Host
To initiate studies on C. albicans genes required for
infection or colonization, preliminary microarray analysis
of gene expression in C. albicans cells recovered from sites of
oral infection was performed. WT C. albicans cells were orally
inoculated into germ-free newborn piglets as described .
Following immunosuppression with cyclosporine A and
methylprednisolone for 7–10 d, the animals developed
dramatic thrush and esosphagitis, and C. albicans plaques
were visible in the esophagus at necropsy. C. albicans cells were
scraped from the tongues of two immunosuppressed, gnoto-
biotic (IGB) piglets and from the esophagus of one of them
and were used for microarray analysis as described in
Materials and Methods. Because the majority of spots
exhibited poor hybridization with samples from the oral
infections, a comprehensive analysis of differential gene
expression could not be performed. However, there were
several candidate genes more highly expressed in samples
taken from infected piglets than in reference laboratory-
To confirm that candidate genes from the microarray
studies exhibited increased expression in mammalian hosts
compared to log phase laboratory-grown cells, quantitative
real-time reverse transcriptase PCR (qRT-PCR) was per-
formed. For the analysis, RNA was prepared from C. albicans
cells scraped from the tongue, esophagus, or roof of the
mouth of two IGB piglets or recovered from the contents of
the large intestine of one IGB piglet. cDNA prepared from
the RNA was used as the template for qRT-PCR amplification
as described in Materials and Methods. Results were
normalized using C. albicans ACT1 (encoding actin) and are
shown relative to the reference sample of log phase,
laboratory-grown cells that were used for the microarrays.
Seven genes that exhibited relatively high expression in C.
albicans cells recovered from the oral cavity or intestinal tract
were identified, including EFH1, YHB5, and ECE1 (Table 1),
which became the subjects of this study. EFH1 encodes a
paralog of the well-studied transcription factor Efg1p .
Efg1p regulates several morphological transitions as well as
Table 1. Gene Designations
Gene NameORF19 DesignationCYGDaDesignation
aComprehensive Yeast Genome Database.
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Self-Regulation of Colonization
Although the fungus Candida albicans commonly colonizes the
human gastrointestinal tract as a commensal, the organism is also
an opportunistic pathogen, responsible for a wide range of
infections in immunocompromised persons. While numerous
studies of infection have been conducted, few studies have analyzed
the commensal state. The studies described here analyze C. albicans
cells colonizing the intestinal tract of immunocompetent mice in the
absence of disease, a model for commensalism. Results showed that
expression of the putative transcription factor Efh1p by cells
colonizing the intestinal tract was relatively high, but paradoxically,
expression of Efh1p was associated with lower colonization. Efh1p
had no detectable effect on the ability of C. albicans to cause lethal
disseminated infection in mice. In contrast, Rbt1p and Rbt4p, two
proteins of poorly defined function required for normal dissemi-
nated infection, were not required for intestinal colonization. These
results argue that the commensal state is distinct from the
pathogenic state and that different factors are important in different
states. Also, the regulation of expression of genes RBT1, RBT4, and
ECE1 during intestinal colonization differed from their well-
characterized regulation during laboratory growth. Further studies
of commensal colonization are needed to understand this important
stage of C. albicans biology.
the expression of hyphal and virulence genes [14,18–21].
However, the absence of Efh1p does not lead to a pronounced
phenotype during laboratory growth , and thus, its
function is not understood. YHB5 (orf19.3710) encodes a
flavohemoglobin; its paralog, YHB1, is induced by growth in
the presence of nitric oxide and is important for resistance to
nitrosative stress [22,23]. YHB5, by contrast, is not induced
during laboratory growth in nitric oxide . ECE1, a gene
that influences adhesion , but whose function is poorly
understood, is highly expressed by cells that are growing in
the hyphal form (filamentous cells lacking constrictions at
their septa) [21,24,25]. ECE1 is also highly expressed during
invasion of host tissue .
In addition, other genes expressed under laboratory
conditions that resulted in increased ECE1 or EFH1 expres-
sion (hyphal growth and post-exponential phase, respectively)
were analyzed. RBT1 and RBT4, genes of poorly understood
function that are expressed in hyphal cells , and SOD3
(orf19.7111.1), encoding a manganese-containing superoxide
dismutase that is expressed in post-exponential phase ,
were studied. Finally, a control housekeeping gene, TEF1
(encoding translation elongation factor 1-alpha), was ana-
lyzed in some experiments.
The qRT-PCR results for the genes of interest are shown in
Figure 1. In C. albicans cells recovered from oral infection, five
of the genes were highly expressed relative to their expression
in log phase laboratory-grown cells (Figure 1A). The sixth
gene, EFH1, was not convincingly upregulated in cells
recovered from oral lesions. In cells recovered from the
piglet intestine, EFH1 and the five other genes showed
relatively high expression (Figure 1B, closed circles). Since
only one piglet intestinal tract sample was studied, these
initial data on gene expression in the intestinal tract were not
definitive. Further studies in mice corroborated the results, as
To compare expression during growth in the host with
expression during growth in laboratory conditions that might
mimic certain host parameters, cells were grown to a post-
exponential phase (rich medium 37 8C, 3 d) or under
conditions that resulted in formation of hyphae (serum-
containing medium, 37 8C), and compared to log phase cells
(rich medium 37 8C). Expression was measured by qRT-PCR
as above. Figure 1C shows that most of the genes of interest
were more highly expressed in post-exponential phase than
in log phase and all genes except for EFH1 were relatively
highly expressed in hyphae. In contrast, expression of the
housekeeping gene TEF1 was not markedly induced under
these conditions. Thus, several genes were relatively highly
Figure 1. Expression of C. albicans Genes
Expression of genes of interest in cDNA preparations was determined by qRT-PCR as described in Materials and Methods. Results were normalized using
actin expression and are expressed relative to a reference sample of laboratory-grown log phase cells.
(A) Gene expression in C. albicans cells recovered from the oral cavities or esophagi of IGB piglets. Closed circles, WT C. albicans cells (SC5314) recovered
from the tongue, roof of the mouth, or esophagus; bars, geometric means.
(B) Gene expression in C. albicans cells recovered from the intestinal tract. Closed circles, WT C. albicans cells (SC5314) recovered from the large intestine
of an IGB piglet; closed triangles, WT (SC5314 or DAY185), or mutant (efg1 cph1 double null) C. albicans cells recovered from the cecum of Swiss Webster
mice; open triangles, WT C. albicans (SC5314) recovered from the ileum of Swiss Webster mice; bars, geometric means.
(C) Gene expression during laboratory growth. White bars, log phase C. albicans grown in rich medium at 37 8C; black bars, post-exponential phase C.
albicans grown in rich medium at 37 8C; grey bars, hyphal C. albicans grown in RPMI-serum at 37 8C; error bars, standard deviation.
(D) Comparison of gene expression in WT C. albicans (DAY185) and in an efg1 deletion mutant grown in RPMI-serum at 37 8C. Cross-hatched bars,
relative ECE1 expression; white bars, relative RBT1 expression; black bars, relative RBT4 expression; error bars, standard deviation.
PLoS Pathogens | www.plospathogens.org December 2007 | Volume 3 | Issue 12 | e184 1868
Self-Regulation of Colonization
expressed during growth within multiple tissues in the host
and in either post-exponential phase or hyphal growth in the
Gene Expression during Intestinal Colonization Is Not
Dependent on Host Immunosuppression
For further studies of the interaction between C. albicans
and a mammalian host, we analyzed gene expression during
colonization of immunocompetent Swiss Webster mice.
Following oral inoculation of antibiotic-treated Swiss Web-
ster mice, commensal colonization persisted for several weeks
as previously described, e.g., [27–29]. No symptoms of disease
were observed in the colonized mice.
To determine whether the genes of interest were expressed
during commensal intestinal colonization, gene expression
was measured by qRT-PCR using RNA prepared from C.
albicans cells recovered from the cecum or ileum of colonized
mice. Expression normalized to ACT1 is expressed relative to
the same reference sample as above. As shown in Figure 1B
(closed triangles), all six genes of interest were expressed at
relatively high levels in C. albicans cells recovered from the
mouse cecum in contrast to the housekeeping gene TEF1.
Expression was very similar to the expression observed in C.
albicans cells recovered from the IGB piglet intestinal tract.
Therefore, the expression of these genes was not dependent
on immunosuppression of the host. In addition, to compare
expression in a different part of the intestinal tract,
expression of three genes was characterized in cells recovered
from the ileum. ECE1 and RBT4 were relatively highly
expressed and RBT1 was slightly increased. In summary, five
of the six genes were relatively highly expressed during
growth within multiple tissues of immunosuppressed and
immunocompetent hosts. EFH1 exhibited a distinct pattern
of expression as it was relatively highly expressed in C. albicans
cells recovered from the intestinal tract but not from sites of
oral infection (esophagus or tongue lesions).
Uncharacterized Regulatory Pathways Control Gene
Expression during Growth in the Intestinal Tract
Because most of the genes of interest were highly expressed
in laboratory-grown hyphae (Figure 1C), it was of interest to
determine the morphology of the colonizing cells. C. albicans
cells expressing yeast-enhanced green fluorescent protein
(yEGFP)  were orally inoculated into mice by gavage.
Three, seven, or 17 days post-inoculation, the contents of the
ileum were recovered, and the C. albicans cells were observed
by fluorescence microscopy. In all samples, the vast majority
of the fluorescent cells (.90% þ/? 3% standard deviation)
exhibited yeast cell morphology (Figure 2). Because the
cecum is anaerobic, it was not possible to visualize GFP in
this organ, so phase contrast microscopy was used to visualize
organisms. As with the ileum, the majority of fungal cells
detected in cecum contents exhibited the yeast-cell morphol-
ogy (71% þ/? 10% standard error of the mean).
Interestingly, cells recovered from the ileum expressed
higher levels of ECE1 and RBT4 than laboratory-grown yeast
cells and these levels were comparable to laboratory-grown
hyphal cells. This observation suggested that the yeast-form
cells recovered from the ileum, rather than the rare hyphal-
form cells, were expressing high levels of ECE1 and RBT4.
Therefore, gene expression during growth within the host
differed from the characterized expression of ECE1 and RBT4
under laboratory conditions.
To compare gene expression during laboratory growth and
host growth further, the role of the morphogenesis-regulating
transcription factor Efg1p was studied. In the laboratory,
expression of ECE1, RBT1, and RBT4 in hyphae is dependent
on Efg1p (Figure 1D and [31,32]). Expression is similarly
reduced in a double mutant lacking a second partially
redundant transcription factor, Cph1p  and Efg1p
(unpublished data and ). Therefore, the effect of deletion
of both EFG1 and CPH1 on expression of these three genes
during growth in the intestinal tract was studied. Strain
CKY138 (efg1 cph1 double null mutant) was orally inoculated
into mice by gavage. RNA was prepared from cells recovered
from the intestinal tract and expression of ECE1, RBT1, and
RBT4 was measured by qRT-PCR. Unexpectedly, double null
mutant C. albicans cells recovered from the intestinal tract
expressed ECE1, RBT1, and RBT4 (Figure 1B). Thus, there was
substantial Efg1-, Cph1p-independent expression of ECE1,
RBT1, and RBT4 during growth within the intestinal tract.
These observations demonstrate that an uncharacterized
regulatory pathway(s) controls the expression of these genes
during growth in the intestinal tract, underscoring the
importance of studying C. albicans physiology during growth
within a host.
Expression of EFH1 Results in Reduced Colonization of the
Five of the six genes of interest were expressed at elevated
levels in all samples recovered from the host, while EFH1
Figure 2. C. albicans Cells Colonizing the Murine Intestinal Tract Are
Predominantly in the Yeast Form
(A and B) Contents of the ileum of mice inoculated with C. albicans strain
CKY368, WT, GFP-expressing, day 3 post-inoculation (A) or strain CKY374,
efh1 null mutant, GFP-expressing day 13, post-inoculation (B). Fluo-
rescence micrographs show the morphology of yeast-form cells (A) or
hyphal-form cells (B).
(C) Quantitation of cellular morphology.
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Self-Regulation of Colonization
expression was elevated in cells recovered from the intestinal
tract but not from the tongue or esophagus. To determine
whether EFH1 performs a function during colonization of the
intestinal tract, efh1 null mutants and EFH1 reconstituted
strains were constructed.
As previously reported , deletion of EFH1 did not result
in defects in growth under laboratory conditions. Hyphal
formation by the mutant strain was also normal and the efh1
null mutant was indistinguishable from WT in post-expo-
nential phase stress resistance (heat shock at 55 8C or
menadione treatment; unpublished data). The mutant strain
grew well anaerobically in defined medium  (I. Soltero
and C. A. Kumamoto, unpublished data). Therefore, as noted
previously , the efh1 deletion appeared to have minimal
effects on the physiology of laboratory-grown cells.
When WT C. albicans, efh1 null mutant, or EFH1 recon-
stituted null mutant cells were orally inoculated by gavage
into immunocompetent mice, intestinal tract colonization
was established. As shown in Figure 3, WT C. albicans
colonized the intestinal tract and was detectable in the
contents of the cecum up to 3 wk post-inoculation (Figure 3A,
closed circles). Colonization was also detectable in the ileum,
stomach, and fecal pellets (Figure 3B and 3C); analysis of
these samples from three individual mice revealed a
correlation in colonization levels in different organs (Figure
To determine the effect of coprophagy on colonization, an
uninoculated mouse was introduced into a cage with three to
four inoculated mice, 24 h post-inoculation. Fecal pellets
from the uninoculated mouse were initially positive for C.
albicans, but the levels of C. albicans declined very rapidly, and
8 d post-introduction into the cage were at least 50-fold
below the geometric mean for the inoculated mice. There-
fore, consistent with previous studies , the contribution to
C. albicans titers due to coprophagy was minimal at later time
Surprisingly, two independently isolated efh1 null mutants
colonized the murine intestinal tract at higher levels (Figure
3A, blue triangles) than WT C. albicans (Figure 3A, red circles).
In the cecum at day 21 post-inoculation, the geometric mean
for the efh1 null mutant was 100-fold higher than that for WT
C. albicans (p , 0.000005 by t test). For both the efh1 null
mutant and WT strains, C. albicans associated with the cecum
wall represented a small fraction of the total cecum-
associated C. albicans. Analysis of C. albicans in fecal pellets
demonstrated that, initially, colonization by the mutant was
Figure 3. Deletion of EFH1 Alters Murine Intestinal Colonization
WT, efh1 deletion mutant, or EFH1 reconstituted null mutant were orally inoculated by gavage into Swiss Webster mice. At various days post-
inoculation, the amounts of C. albicans in fecal pellets and in organs of the intestinal tract were measured.
(A) CFUs per gram of cecum contents from mice sacrificed on the indicated days post-inoculation. Each symbol represents a sample from a different
mouse. (Composite results from several experiments.) Red circles, WT C. albicans strain DAY185 (23 mice); blue triangles, efh1 deletion mutant strain
CKY366 (24 mice); black diamonds, EFH1 reconstituted strain, CKY373 (ten mice); open symbols, no colonies detected; bars, geometric means. p-Value
was determined using the t test with log transformed data. ** indicates p , 0.000005.
(B) CFUs per gram of fecal pellet. Mice were sampled repeatedly and each symbol represents a sample from a different mouse. Different numbers of
mice are shown on different days because some mice were sacrificed earlier and some mice were sampled on different days. Red circles, WT C. albicans
strain DAY185 (ten mice); blue triangles, efh1 deletion mutant strain CKY366 (12 mice); black diamonds, EFH1 reconstituted strain, CKY373 (seven mice);
open symbols, no colonies detected; bars, geometric means. p-Value was determined using the t test with log transformed data. * indicates p , 0.0003.
(C) Correlation between CFUs measured in several organs and in fecal pellets. Light blue diamonds, fecal pellets (labeled F); pink triangles, cecum
contents (labeled C); open circles, ileum contents (labeled I); squares, stomach contents (labeled S). CFU/gm of cecum contents, stomach contents, and
fecal pellets correlated well, while the CFU/gm ileum was more variable.
(D) Mice were colonized with WT strain DAY185 (red circles) or efh1 null mutant CKY366 (blue triangles) or were not inoculated with C. albicans (not
shown). Mice were sacrificed on day 21 post-inoculation, CFUs from cecum contents were determined by plating, and C. albicans DNA in cecum
contents was quantified by qPCR as described in Materials and Methods. The correlation between CFUs in 70 mg of pelleted cecum contents (x-axis)
and DNA (in arbitrary units) determined by qPCR (y-axis) is shown. Each symbol represents a different mouse.
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Self-Regulation of Colonization
similar to colonization by WT C. albicans (Figure 3B) but at
later time points, e.g., day 21 post-inoculation, colonization
by the efh1 null mutant persisted at higher levels than the WT
strain (Figure 3B, p , 0.0003 by t test). Enhanced colonization
was also observed in the ileum and stomach (Figure 3C and
As a control to show that the differences in colony forming
units (CFUs) recovered from WT or efh1 mutant–containing
ceca truly reflected differences in the numbers of fungal cells
rather than preferential recovery of mutant cells, an
alternative method of quantification was used. DNA was
extracted from cecum contents and the amount of C. albicans
genomic DNA was determined by quantitative PCR (qPCR).
The results showed that the amounts of C. albicans DNA
recovered from cecum contents containing WT or efh1 null
mutant cells (in arbitrary units) correlated with the CFUs
determined by plating (Figure 3D), and the average ratio of
the amount of DNA/CFU was the same for the WT and efh1
null mutant strains. Therefore, differences in CFUs reflected
differences in the numbers of colonizing C. albicans.
To demonstrate that the enhanced colonization reflected
the absence of Efh1p, EFH1 was added back to the deletion
mutant under control of a strong promoter to ensure
expression of the reintroduced gene. Introduction of the
ectopically controlled EFH1 to the mutant strain resulted in
reduced colonization (Figure 3A, black diamonds), indicating
that the level of EFH1 in the strain determined colonization
levels. For unknown reasons, introduction of EFH1 at its
native locus did not result in full complementation (unpub-
lished data), as has been observed by others for other genes,
e.g., . These studies demonstrate that in the absence of
Efh1p, colonization of the intestinal tract was enhanced.
Thus, paradoxically, expression of C. albicans EFH1 during
commensal colonization of the intestinal tract resulted in
To observe the morphology of efh1 null mutant cells, GFP-
expressing efh1 null mutants were orally inoculated into mice
by gavage. On day 3 or day 13 post-inoculation, ileum
contents were collected and organisms were visualized by
observing green fluorescence. As observed for WT C. albicans,
the vast majority (.91% þ/? 1% standard deviation) of cells
exhibited yeast-form morphology (Figure 2).
To detect dissemination from the intestinal tract in mice,
the kidneys, liver, and spleen were homogenized and
cultured. With both efh1 null mutant and WT, all samples
were either negative or contained very few organisms (Table
2). Therefore, there was no evidence of high-level coloniza-
tion of deep organs, indicating that C. albicans was not
escaping from the intestinal tract.
Colonization of the tongue of mice was analyzed to
determine whether a significant level of oral candidiasis was
occurring. Colonization was generally not detectable,
although an occasional mouse exhibited below 103CFU/gm
tongue tissue (Table 2). No consistent differences in
colonization by WT and mutant organisms were observed.
Therefore, there was no evidence of either mucosal or
systemic disease in these immunocompetent mice.
To determine the levels of residual bacteria remaining
after antibiotic treatment, ileum and cecum homogenates
were cultured under aerobic or anaerobic conditions on rich
media. The ranges of bacteria levels were very similar for
uninoculated mice and for mice inoculated with all of the C.
albicans strains described above (unpublished data).
EFH1 Overexpression Reduces Intestinal Colonization
Since reduction of Efh1p by deletion resulted in enhanced
colonization, the effect of increased expression of Efh1p was
tested using an EFH1þstrain carrying a third copy of EFH1
expressed from the strong ADH1 promoter. The growth rate
of the EFH1 overexpressing strain was close to WT during
laboratory growth in CM medium at 37 8C (WT doubling time
78 min; overexpressing strain, 82 min), and high levels of
EFH1 transcript were produced under these conditions
(unpublished data). In rich medium, the overexpressing strain
produced yeast-form cells while in certain minimal media the
mutant produced pseudohyphae, consistent with previous
When the overexpressing mutant was orally inoculated into
mice by gavage, colonization was initially similar to that of
WT C. albicans (Figure 4). Subsequently, however, colonization
by the overexpressing strain declined more rapidly than
colonization by the WT strain, and at days 18 and 21 post-
inoculation, the geometric mean for the overexpressing
strain was more than 100-fold lower than that of the WT
strain (p , 0.002 using t test). This result is consistent with
that of the EFH1 reconstituted null mutant (Figure 3).
Therefore, high expression of EFH1 attenuated colonization,
while deletion of EFH1 resulted in enhanced colonization,
demonstrating that Efh1p is a regulator of the level of
colonization during growth of C. albicans in the murine
Effects of EFH1 on Disease
In piglet oral lesions, expression of EFH1 was low relative
to its expression in cells growing within the intestinal tract.
These findings suggest that when infection occurs, the
negative effects of EFH1 on host colonization are relieved
by lowering EFH1 expression. Therefore, ectopic EFH1
overexpression might reduce the ability of C. albicans to cause
disease. To test this model, the behavior of an EFH1
overexpressing strain was analyzed in two different animal
In immunocompromised patients, C. albicans causes mu-
cosal infections, such as OPC. Paralleling the susceptibility of
AIDS patients to OPC, mice lacking T cells show an enhanced
susceptibility to oral colonization by C. albicans . To
determine whether EFH1 overexpression would influence
Table 2. Organ Colonization
CKY366 (efh1 null)
CKY373 (EFH1 reconstituted)
aPositive for colonization/total mice tested (composite of four or five experiments).
bPositive: 13 CFU/gm.
cRange of positives: 160–430 CFU/gm.
dRange of positives:420–6,900 CFU/gm.
eRange of positives: 42–94 CFU/gm.
fPositive: 80 CFU/gm.
gPositive: 280 CFU/gm.
hPositive for colonization/total mice tested (composite of two experiments).
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Self-Regulation of Colonization
colonization in the oral cavities of immunocompromised
mice, competition experiments were performed, in which
mixtures of EFH1 overexpressing and genetically marked WT
C. albicans cells were inoculated directly into the oral cavities
of athymic mice by swabbing. The marked WT strain carried
the SAT1 nourseothricin resistance gene  under control of
the maltase promoter. To monitor the ability of the two
strains to persist in the oral cavity relative to one another, the
oral cavities were swabbed at various times post-inoculation,
and the ratio of the two strains was determined by replica
plating. The competitive index (CI) was determined by
dividing the ratio of NouSand NouRstrains at a particular
time point by the ratio in the inoculum.
When unmarked WT and marked WT strains were mixed
and inoculated, the geometric means for the CI remained
above 1 throughout the time course, indicating a slight
competitive advantage for the unmarked WT strain (Figure
5). For the competition between the NouSEFH1 over-
expressing strain and the NouRWT strain, the CI was close
to 1 on day 1 post-inoculation, but declined thereafter,
demonstrating that the EFH1 overexpressing strain exhibited
a competitive disadvantage relative to the WT strain (Figure
5). The difference in CI for WT and EFH1 overexpressing
strain was statistically significant (p , 0.01 by t test). At longer
times, few colonies were obtained, precluding the accurate
measurement of ratios. Following 24 h of laboratory growth
in CM medium at 37 8C, the CI for the overexpressing strain
relative to the marked WT was 1.1 (average of two
determinations), showing a similar growth rate for the strains
under these conditions. Therefore, these data show that
forced high expression of EFH1 reduces colonization in the
oral cavity of immunodeficient mice. In immunocompetent
BALB/c mice, oral colonization was rapidly lost (unpublished
data), and it was not possible to measure ratios.
To determine whether EFH1 was important in other host
niches, the ability of the efh1 null mutant and EFH1
overexpressing strain to cause lethal infection in a dissemi-
nated candidiasis model was tested. When inoculated intra-
venously in mice, both the efh1 null mutant and the EFH1
overexpressing strain retained the ability to cause lethal
infections. The mean survival time for mice inoculated with
WT C. albicans was 5 6 3 d (23 mice). For the efh1 null mutant,
the mean survival time was 5 6 3 d (16 mice) and for the
EFH1 overexpressing strain, the mean survival time was 7 6 3
d (seven mice) (composite results from at least two experi-
ments). Therefore, both the efh1 null mutant and the EFH1
overexpressing strain were virulent in this model.
rbt1 and rbt4 Mutants Colonize the Murine Intestinal Tract
at WT Levels while the ece1 Mutant Is Attenuated
The ability of mutants lacking some of the other genes of
interest to colonize the intestinal tract of mice was analyzed.
The rbt1 null mutant (Figure 6A), rbt4 null mutant (Figure 6A),
and yhb1 yhb5 double null mutant (Figure 6B) colonized the
intestinal tract at close to WT levels. In contrast, the ece1
mutant exhibited an attenuated colonization phenotype,
which was reversed when the WT ECE1 gene was added back
to the mutant strain (Figure 6C). Therefore, two of the six
genes tested in this study that were relatively highly expressed
during growth in the intestinal tract, EFH1 and ECE1,
influenced the ability of C. albicans to colonize.
In this communication, several genes that were relatively
highly expressed during growth within a host (host-growth
Figure 4. Attenuated Colonization of Mice by an EFH1 Overexpressing
Cells of WT strains CKY363 or DAY185, efh1 deletion mutant strain
CKY366, and EFH1 overexpressing strain CKY364 were orally inoculated
by gavage. At various days post-inoculation, fresh fecal pellets were
recovered from inoculated mice and the amount of C. albicans per gm
was measured. Red circles, CKY363 or DAY185 (WT) (seven mice); blue
triangles, efh1 deletion mutant strain CKY366 (six mice); dark green
diamonds, CKY364 (EFH1 overexpressing) (eight mice); bars, geometric
means. Composite results of two experiments are shown. Different
numbers of mice are shown on different days because some mice were
sacrificed earlier and some mice were sampled on different days.
Figure 5. EFH1 Overexpression Reduces Colonization of the Oral Cavity
in Immunodeficient Mice
Cells of strains CKY363 (WT) or CKY364 (EFH1 overexpressing, OE) were
mixed in a 1:1 ratio with strain RMIS1 (NouRmarked WT) and introduced
into the oral cavities of nude mice by swabbing. On various days post-
inoculation, the oral cavities were sampled by swabbing. Swabs were
rubbed on YPD SA plates and the colonies were replica-plated to
YPSnourseothricin to determine the ratio of NouSto NouRcolonies. The
CI is defined as the ratio of NouS/NouRcolonies at time x, divided by their
ratio in the inoculum. Each symbol represents a sample taken from an
individual animal. Composite results of two experiments are shown. Red
circles, CKY363 (WT) mixed with RMIS1 (seven mice); dark green
diamonds, CKY364 (EFH1 overexpressing) mixed with RMIS1 (six mice).
Day ?5 includes samples taken at either day 5 or day 6. Samples that
yielded very few colonies (,10) were not included on the graph. A
statistically significant difference (p , 0.01 by t test) was observed at day
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Self-Regulation of Colonization
genes) were characterized. The host-growth genes differ in
their importance for intestinal colonization and systemic
virulence demonstrating that different factors control host-
pathogen interactions in different sites. The genes RBT1 and
RBT4 are required for C. albicans to cause lethal infection
following intravenous inoculation and for invasive infection
of the cornea . However, these genes are not required for
colonization of the intestinal tract. In contrast, the C. albicans
EFH1 gene regulates intestinal colonization but not systemic
infection. Thus, some genes are required in one niche but not
in another. Intestinal colonization is an essential stage in the
life cycle of C. albicans because the organism is not thought to
have an environmental reservoir. Therefore, optimal intesti-
nal colonization is crucial for survival of the organism, and it
is not surprising that the organism possesses genes that
regulate colonization in this niche.
EFH1 was relatively highly expressed by C. albicans cells
growing in the mammalian intestinal tract, but paradoxically,
expression of EFH1 was associated with lower colonization.
Ectopic overexpression of EFH1 resulted in very poor
colonization; this latter finding probably reflects a true
difference in colonization, because for in vitro–grown EFH1
overexpressing cells, the ratio of DNA content (determined
by qPCR) to CFUs (measured by plating) was less than 2-fold
less than the ratio obtained for WT C. albicans (unpublished
data). In this niche, therefore, expression of EFH1 had a
negative effect on population size. Expression of Efh1p may
affect the interactions between colonizing C. albicans and the
cells of the intestinal tract, resulting in changes such as
altered adherence or altered signaling to the immune system.
By reducing colonization, expression of EFH1 may also
reduce the likelihood of C. albicans infection, favoring
commensal colonization as opposed to candidiasis.
The effects of Efh1p on colonization are reversible, because
expression of EFH1 was regulated during growth within the
host (lower expression in cells recovered from OPC lesions
than in cells recovered from the intestinal tract). Therefore,
the negative effects of Efh1p that reduce colonization are
inactivated during oral infection. Since forced expression of
EFH1 reduced colonization of the oral cavity in an
immunodeficient host, repression of EFH1 expression during
active infection may be an important step in the progression
from benign colonizer to active invader on mucosal surfaces.
Since EFH1 encodes a putative transcription factor, its
effects on colonization are probably indirect. Most likely,
deletion of EFH1 alters the expression of other genes, some of
which directly influence colonization. Under laboratory
conditions, EFH1 did not strongly affect the expression of
the collection of genes studied in this report (unpublished
data). However, in previous studies, a laboratory-grown EFH1
overexpressing strain was shown to have altered expression of
several cell surface proteins , which may play roles in
The genes studied here were relatively poorly expressed in
laboratory-grown log phase cells but showed expression in
laboratory-grown post-exponential phase cells, suggesting
that during growth within a host, C. albicans cells express some
features of post-exponential phase cells. Numerous bacterial
genes that play important roles governing host-pathogen
interactions are expressed in post-exponential phase. For
example, expression of virulence factors by organisms such as
Bacillus anthracis , Staphylococcus aureus , Helicobacter
Figure 6. rbt1 and rbt4 Mutants Colonize the Murine Intestinal Tract at
Cells of WT strains DAY185 or F2U, rbt1 deletion mutant strain BCa 7–4,
rbt4 null mutant strain BCa 11–3, ece1 null mutant CAW19–1, CKY362
(ECE1 reconstituted), and CKY376 (yhb1 yhb5 double null mutant) were
orally inoculated by gavage. At various days post-inoculation, fresh fecal
pellets were recovered from inoculated mice and the amount of C.
albicans per gm was measured. Composite results of two experiments
are shown. Different numbers of mice are shown on different days
because some mice were sacrificed earlier and some mice were sampled
on different days.
(A) Red circles, DAY185 or F2U (WT) (six mice); green triangles, rbt1
deletion mutant strain BCa 7–4 (six mice); blue diamonds, rbt4 deletion
mutant strain Bca 11–3 (six mice); bars, geometric means.
(B) Red circles, DAY185 (WT) (six mice); green triangles, yhb1 yhb5 double
null mutant strain CKY376 (six mice); bars, geometric means.
(C) Red circles, DAY185 or F2U (WT) (six mice); green triangles, ece1
deletion mutant strain CAW19–1 (six mice); black diamonds, ECE1
reconstituted mutant strain CKY362 (four mice); bars, geometric means.
Open symbols, no colonies detected. Day ? 14 indicates samples taken
on day 14 or day 15.
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Self-Regulation of Colonization
pylori , Legionella pneumophila , or Salmonella  occurs
in post-exponential phase. Therefore, expression of post-
exponential phase genes during host interaction is a common
theme for many bacterial pathogens and for the fungus C.
In the laboratory, expression of ECE1, RBT1, and RBT4 is
linked to hyphal morphogenesis. However, in cells growing
within the intestinal tract, expression of these genes does not
depend on Efg1p and most likely occurs in yeast cells. ECE1,
RBT1, and RBT4 were also found to be expressed in efh1 null
mutants recovered from the cecum of mice (unpublished
data). Therefore, the pathway(s) responsible for the expres-
sion of these genes during growth within the host is
uncharacterized. Since the genes show relatively high
expression during post-exponential phase in the laboratory,
there may be a post-exponential phase regulator(s) that is
responsible for their expression in the host. Several regu-
lators required for normal viability in post-exponential phase
have been recently described . Deeper understanding of
the biology of post-exponential phase cells may reveal C.
albicans activities that are important for colonization and
During colonization, the population size of a microorgan-
ism reflects a balance between external forces that limit the
population, such as the effects of the host immune system,
and intrinsic factors, such as the ability of the organism to
increase in number. The ability of commensal C. albicans to
regulate its own population size through reversible expres-
sion of a negative regulator of colonization adds another
layer of regulation to the interactions that take place between
host and colonizer. The combined effects of these regulatory
interactions maintain the balance between healthy coloniza-
tion and disease.
Materials and Methods
Strains. C. albicans strains are listed in Table 3. All C. albicans strains
were derived from the WT clinical strain SC5314 , using the
following genetically marked derivatives: CAI-4 , BWP17 ,
RM1000#2 , or SN100 .
Strain CKY366, the efh1 null mutant strain, was constructed by
transformation of BWP17 with constructs encoding HIS1 or ARG4
flanked by 600 bp of sequence upstream of the EFH1 ORF and 542 bp
of sequence downstream of the EFH1 ORF. The fragment was excised
from the plasmid backbone by digestion with BslI and BsmFI for
efh1D::ARG4 or with NotI sites that were introduced at the ends of the
EFH1 sequences for efh1D::HIS1.
The efh1 null mutant was transformed with pRC3915  to restore
URA3 prototrophy or with pCK73 to introduce EFH1 under control
of the ADH1 promoter.
To construct an EFH1 overexpressing strain, the EFH1þstrain
SN100 (his1/his1) was transformed with pCK74 encoding EFH1 under
control of the ADH1 promoter and carrying selectable marker HIS1.
yEGFP-  expressing EFH1þand efh1 null mutant strains were
constructed by transforming RM1000 Hisþor the efh1 null mutant
with pDRG-GFPS6, encoding yEGFP under control of the DRG1
Strain CKY375, the yhb5 null mutant strain, was constructed by
transformation of BWP17 with constructs encoding HIS1 or ARG4
flanked by 496 bp of sequence upstream of the YHB5 ORF and 355 bp
of sequence downstream of the YHB5 ORF. The desired fragments
were excised from their plasmid backbones by digestion with TseI
and BslI for yhb5D::ARG4 or with NotI for yhb5D::HIS1. Strain
CKY376, the yhb1 yhb5 double null mutant strain, was constructed
by transformation of the yhb5 deletion mutant with a construct
encoding the SAT flipper  with 750 bp of sequence upstream of
the YHB1 ORF and 490 bp of sequence downstream of the YHB1 ORF.
Nourseothricin-resistant (NouR) transformants were screened ini-
tially by PCR. Positive candidates were plated on sucrose-containing
medium to induce expression of the FLP recombinase and replica-
plated to nourseothricin medium. NouScolonies were purified and
Table 3. Candida albicans Strains
StrainGenotype Source or Reference
WT clinical isolate
SC5314 ura3D::kimm434/ ura3D::kimm434
SC5314 ura3D::kimm434 / ura3D::kimm434 his1::hisG/his1::hisG
SC5314 his1/his1 URA3/ ura3D::kimm434
SC5314 his1/his1 ura3D::kimm434/ ura3D::kimm434
SC5314 HIS1/his1 ura3D::kimm434/ ura3D::kimm434
ura3D::kimm434/ ura3D::kimm434 HIS1::his1::hisG/ his1::hisG ARG4::URA3::
CAI-4 URA3/ ura3D::kimm434
CAI-4 efg1::hisG/efg1::hisG (ade2::pDBI52(URA3))
CAI-4 efg1::hisG/efg1::hisG cph1::hisG/cph1::hisG (ade2::pDBI52(URA3))
CAI-4 rbt1D::hisG/rbt1D::hisG-URA3-hisG ura3D/ura3D
CAI-4 rbt4D::hisG/rbt4D::hisG-URA3-hisG ura3D/ura3D
CAI-4 ece1D::hisG::I-SceI/ece1D::hisG::I-SceI ura3D/URA3
BWP17 efh1D::ARG4/efh1D::HIS1 leu2::pRC3915 (URA3)
BWP17 efh1D::ARG4/efh1D::HIS1 adh1::pCK73(PADH1-EFH1, URA3)
BWP17 yhb5D::ARG4/yhb5D::HIS1 leu2::pRC3915 (URA3)
BWP17 yhb5D::ARG4/yhb5D::HIS1 yhb1D::FRT/yhb1D::FRT ura3D/URA3
SN100 adh1::pCK75 (PADH1, HIS1 URA3)
SN100 adh1::pCK74 (PADH1-EFH1, HIS1 URA3)
BWP17 efh1D::ARG4/efh1D::HIS1 DRG1::pDRG-GFPS6
RM1000#2 HIS1/his1 (ade2::iSAT)
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Self-Regulation of Colonization
subjected to a second round of transformation as above and null
mutants were identified by Southern blot analysis. To generate URA3
prototrophs, the strains were transformed with a URA3þfragment
from plasmid pET16 .
The ece1 null mutant CAW19–1 was kindly provided by B. Fonzi
(Georgetown University) and the rbt1 and rbt4 null mutants (BCa 7–4
and Bca 11–3, respectively) were kindly provided by S. Johnson
To construct a genetically marked WT strain, plasmid iSAT was
digested with BsgI and integratively transformed into strain
RM1000#2, Hisþ. The resultant strain (RM1000 iSAT) exhibits
resistance to nourseothricin when grown on plates containing
sucrose but poorer resistance on plates containing glucose. The
iSAT construct was used because expression from the maltase
promoter was expected to be low during growth within a host,
minimizing possible deleterious effects due to expression of the
heterologous SAT1 gene.
Media and growth conditions. Standard rich media were YPD (1%
yeast extract, 2% peptone, 2% glucose) or YPS (1% yeast extract, 2%
peptone, 2% sucrose). Minimal dropout media (lacking uracil,
histidine, arginine, or combinations) were as described previously
. RPMI 1640 (Sigma) with 10% bovine serum was used to promote
hyphal morphogenesis. Nourseothricin (200 lg/ml) was used as
described . For plating contents of the intestinal tract, YPD agar
medium supplemented with 50 lg/ml ampicillin and 100 lg/ml
streptomycin (YPD SA) was used. For competition experiments,
colonies on YPD SA were replica-plated to YPsucrose supplemented
For culturing bacteria from the intestinal tract, BHIS medium was
used (3.7% Difco brain heart infusion broth, 0.5% yeast extract,
0.0015% hemin, 0.2% agar), and plates were incubated aerobically or
anaerobically at 37 8C.
For gene expression studies, reference cells were grown in YPS
liquid medium at 34 8C in log phase. For the experiments shown in
Figure 1C and 1D, cells were grown in YPD liquid medium at 37 8C in
log phase, or in YPD liquid medium at 37 8C for 3 d (post-exponential
phase), and, to stimulate hyphal morphogenesis, in RPMI-10% bovine
serum medium for 4–6 h at 37 8C.
Plasmids. Plasmid pCK73 was constructed by amplifying the EFH1
ORF with primers VEC53F and VEC53R. The resulting fragment was
digested with BglII and XhoI and cloned onto BglII, XhoI-digested
pYB-ADH1pt . Plasmid pCK74 and pCK75 were constructed by
amplifying the C. albicans HIS1 gene with primer AHISF3 and
AHISR3, digesting the resultant fragment with AhdI and NheI
cloning onto XcmI,XbaI-digested pCK73 or pYB-ADH1pt, respec-
For gene deletions, efh1D::ARG4, efh1D::HIS1, yhb5D::HIS1,
yhb5D::ARG4, and yhb1D::SAT flipper constructs were produced in
several steps. First, EFH1 sequences were amplified using primers
pairs 53A and 53B2 or 53C2 and 53D2 followed by overlap PCR. The
1.1-kb fragment with EFH1 upstream and downstream sequences
flanking a unique PacI site with NotI sites on the ends was TOPO-
cloned onto pYES2.1/V5-His-TOPO (Invitrogen) using manufac-
turer’s protocols. The YHB5 locus fragment was amplified with
primers 44KOF1 and 44KOR1 and the YHB1 locus fragment was
amplified with primers YHB1F1 and yhb1R1; the fragments were
TOPO-cloned as above.
To generate gapped or linearized plasmids, the efh1 plasmid was
digested with PacI, the YHB5 plasmid with PacI, and the YHB1
plasmid with HindIII. HIS1 and ARG4 markers were amplified from
pGEM-HIS or pRS-ARG4DSpeI  with primer pair 53MF1 and
53MR1 (for EFH1 knockout constructs) or 44MF1 and 44MR1 (for
YHB5 knockout constructs). The SAT1 flipper  was amplified with
primers HB1NF1 and HB1NR1 for the YHB1 knockout construct.
Digested plasmids were cotransformed with appropriate PCR
products into Saccharomyces cerevisiae strain EGY40 (MATa ura3–1
his3–11 trp1–1 leu2–3,112) . Homologous recombination gener-
ated the desired constructs.
To construct pDRG-GFPS6, the DRG1 promoter region  was
amplified with primers XC3 and XC4 and cloned onto plasmid
pXC31, a vector containing BamHI, NsiI, SalI, and EcoRV sites
upstream of a promoterless yEGFP gene that was fused to the 39 UTR
of ACT1 in pNUB1 (pNEB193 from New England Biolabs, with C.
albicans URA3 ).
To construct plasmid iSAT (encoding SAT1 under control of the
maltase promoter), the SAT1 ORF was amplified with primers SATEC
and SATR1 from pSFS2 . The 39 UTR of ACT1 was amplified with
primers SATAFU and ACT3x and fused to the SAT1 fragment by
overlap PCR. The resultant fragment was cloned onto vector pDBI52,
generating a plasmid carrying SAT1 under control of PMALwith a
fragment of C. albicans ADE2 for integration and the C. albicans URA3
gene as a selectable marker.
Animal models. (1) Two germ-free piglets were inoculated orally
with 109CFUs of C. albicans strain SC5314 and treated with 25 mg/kg
body weight methylprednisolone and 15 mg/kg body weight cyclo-
sporine daily as described previously . Seven or 10 d post-
inoculation, a piglet was sacrificed and organs were dissected and
frozen in RNALater (Ambion) at ?80 8C.
(2) Female Swiss Webster mice (18–20 g) were treated with
tetracycline (1 mg/ml); streptomycin (2 mg/ml) and gentamycin (0.1
mg/ml) added to their drinking water throughout the experiment
beginning 4 d prior to inoculation. C. albicans cells were grown for 24
h at 37 8C in YPD liquid medium, washed twice with PBS, counted,
and adjusted to 2.53108cells/ml. All strains were prototrophic. Mice
were inoculated by gavage with 53107C. albicans cells in 0.2 ml using
a feeding needle. Colonization was monitored by collecting fecal
pellets (produced within 10 min prior to collection) at various days
post-inoculation and measuring C. albicans concentrations in the
pellets by plating homogenates on YPD SA plates. Mice were
sacrificed on various days post-inoculation and C. albicans concen-
trations in cecum contents, stomach contents, and homogenates of
ileum, kidneys, liver, spleen, and tongue were measured by plating on
YPD SA plates. Composite results from at least two experiments are
(3) Intravenous inoculation of female CF1 mice (18–20 g; Charles
River Laboratories) with 13106C. albicans cells via the lateral tail vein
was conducted as previously described . Mice were observed twice
daily after infection with C. albicans and were sacrificed when
(4) Competition experiments: To generate a marked WT strain,
strain RM1000#2 Hisþwas transformed with plasmid iSAT, resulting
in a prototrophic strain showing inducible resistance to nourseo-
thricin. This genetic marker was used because it was expected that the
maltase promoter would only weakly express the heterologous SAT1
gene during growth of C. albicans in the mouse, thereby minimizing
any potential deleterious effects due to SAT1 expression.
For direct inoculation of the oral cavity by swabbing, the
procedure was based on the previous work of Farah et al.  with
minor modifications. Briefly, female BALB/c nude mice (5–7 wk, NCI)
were given antibiotic water as described above. For inoculation, 1 3
108cells of C. albicans (mixture of two strains at a ratio of 1:1) were
applied to a swab and introduced directly into the oral cavity.
Colonization was monitored by swabbing the oral cavity and by
collecting fecal pellets. Swabs were rubbed on YPD SA plates to assess
colonization; fecal pellets were homogenized and plated. Intestinal
colonization levels were comparable to those obtained by gavage.
To determine the ratios of the two different strains, the YPD plates
were replica-plated to YPSnourseothricin and the ratio of nourseo-
thricin-sensitive colonies (NouS) to nourseothricin-resistant colonies
(NouR) was determined. At the later time points, some samples
yielded few colonies (,10) and these samples were not included on
the graph. The CI was obtained by dividing the ratio of NouS/NouR
colonies at time x by their ratio in the inoculum.
RNA extraction. Tissues from WT strain SC5314-infected IGB
piglets, 7 or 10 d post-inoculation, were frozen in RNALater
(Ambion) at ?80 8C. For tongues and esophagus, the fungal cell–
containing layer was cut from the tissue. WT C. albicans (SC5314 or
DAY185) or mutant CKY138 (efg1 cph1 double null) were inoculated
into Swiss Webster mice by gavage. Three days post-inoculation, the
contents of the cecum or ileum were recovered and frozen in
RNALater at?80 8C. RNA was extracted from these samples using the
Qiagen MIDI procedures with mechanical disruption using glass-
zirconium beads, protease digestion, and on-column DNase treat-
ment. In some experiments, samples were extracted with TRIzol,
applied to a Qiagen column, and treated with DNase. For control
piglet RNA, tissue was cut from the central portion of the tongue of
an infected piglet and was not expected to contain significant
numbers of C. albicans cells. RNA was extracted from this material as
Reference RNA was extracted from cells grown in YPS medium at
34 8C in log phase, using Qiagen procedures with mechanical
disruption and on-column DNase digestion. This RNA preparation
was used as the reference sample for microarrays and for qRT-PCR.
qRT-PCR. 10 lg of total RNA was converted to cDNA by
incubation with Superscript II Reverse Transcriptase (Invitrogen)
using an oligo dT primer. After incubation for 1 h at 42 8C, RNA was
hydrolyzed and the reaction was stopped by addition of NaOH and
EDTA to 0.16 N NaOH, 0.08 M EDTA, final concentrations. Following
neutralization, cDNA was purified using Qiaquick columns (Qiagen)
as described by the manufacturer, except that sodium acetate (pH 5.2)
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Self-Regulation of Colonization
was added to the PB buffer to ensure an acidic pH. cDNA was
quantitated by absorbance. Purified cDNA was stored frozen.
The following primer pairs (Table 4) were used to detect the
EFH1, AB53F1 and AB53R1; YHB5, AB44F1 and AB44R1; ECE1,
CRT91 and AB9R2; RBT1, RBT1F1 and RBT1R1; RBT4, RBT4F1 and
RBT4R1; SOD3, SODF1 and SODR1; ACT1, ABAF3 and ABAR3.
qRT-PCR was performed using SYBR green Mastermix and an
ABI7700 instrument, according to manufacturer’s protocols. All
reactions were performed in triplicate. Melting curve analysis and/or
agarose gel electrophoresis was performed following the reverse
transcriptase PCR amplification to verify the presence of a single
product. When RNA preparations not treated with reverse tran-
scriptase were used as template, the primers failed to amplify
products, or for SOD3, produced a signal nine cycles later than the
signal obtained with cDNA, demonstrating that the amplified
products reflected transcripts. Amplification of cDNA prepared from
efh1 null or yhb5 null mutants with EFH1 or YHB5 primers,
respectively, yielded no signal. cDNA prepared from piglet RNA
yielded either no signal, or for ECE1, RBT1, and RBT4, a signal that
was detected at least six cycles later than the signal obtained with a
sample containing C. albicans. Therefore, the primers were detecting
authentic C. albicans-derived transcripts.
PCR: Cecum contents were collected, and particulate material,
including fungal cells, was pelleted at 3600g for 7 min and weighed. 70
mg of contents were extracted by bead beating in phenol-chloroform
, followed by chloroform extraction and ethanol precipitation.
The DNA was further purified using Qiagen DNeasy methods with
RNase treatment. To determine the amount of C. albicans DNA, qPCR
was performed in triplicate as above with rDNA primers (Table 4)
designed from nonconserved regions of the rDNA sequence. The
amounts of DNA are expressed in arbitrary units obtained using a
standard curve of purified genomic DNA. The signals obtained from
uninoculated mouse ceca were 100-fold lower than the geometric
mean for WT C. albicans inoculated mouse ceca.
Microarrays. Labeled cDNA was prepared by reverse transcription
of RNA with Superscript II Reverse Transcriptase (Invitrogen) using
oligo-dT priming and Cy3- or Cy5-labeled dUTP (NEN). After
incubation for 1 h at 42 8C, RNA was hydrolyzed and the reaction
was stopped by addition of NaOH and EDTA to 0.16 N NaOH, 0.08 M
EDTA, final concentrations. Following neutralization, cDNA was
purified using Qiaquick columns (Qiagen) as described by the
manufacturer, except that sodium acetate (pH 5.2) was added to
the PB buffer to ensure an acidic pH. Dye incorporation was
quantitated by absorbance. Cy3-labeled sample (40–60 pmoles) and
Cy5-labeled reference (20 pmoles) were combined. Labeled sample
cDNA was mixed with reference cDNA from log phase, laboratory-
grown cells, and hybridized to C. albicans microarrays (version 5.1)
containing 6,014 PCR fragments representing 91% of the ORFs in the
C. albicans genome, as described previously . Arrays were
hybridized and scanned using Quantarray , and the results were
analyzed visually or using Excel.
Table 4. Primers
Primer Type Primer NamePrimer Sequence
qRT-PCR primers RBT4F1
gtactagattttcat taa ttggatcat tcctatcta aagata taccctcactaaagggaacaaaagc
Marker amplification primers
PLoS Pathogens | www.plospathogens.orgDecember 2007 | Volume 3 | Issue 12 | e1841876
Self-Regulation of Colonization
Fluorescence microscopy. Swiss Webster mice were inoculated with
yEGFP-expressing C. albicans by gavage as above. At various days post-
inoculation, mice were sacrificed and the contents of the ileum were
recovered. Samples were filtered through 35-lm nylon mesh (Small
Parts) and the filtrate was concentrated by centrifugation in an
Eppendorf centrifuge for 1 min at maximum speed. Samples were
observed using an Olympus BX60 microscope with GFP (excitation
460–490 nm, emission 515–700 nm) and YFP filters (excitation 500/20
nm, emission 535/30 nm) and photographed with the 603 objective.
We thank Daniel Dignard, Donna Akiyoshi, Lauren Logsden, Jenifer
Coburn, Tom Volkert, Jessica Pierce, Eric Rubin, Peter Cheslock,
Perry Riggle, Igor Bruzual, Paola Zucchi, Marcelo Vinces, and Ralph
Isberg for stimulating discussions or help with techniques and Bill
Fonzi and Sandy Johnson for kind gifts of strains. We are also grateful
to Andrew Camilli for careful review of the manuscript. This is NRC
Author contributions. CAK conceived and designed the experi-
ments. SJW, AR, PL, DN, AB, ST, and CAK performed the experi-
ments. SJW, AR, PL, DN, AB, JM, and CAK analyzed the data. MW and
JM contributed reagents/materials/analysis tools. CAK wrote the
Funding. This research was supported by National Institutes of
Health grant AI038591 from the National Institute of Allergy and
Infectious Diseases to CAK.
Competing interests. The authors have declared that no competing
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