Identifying infection-associated genes of Candida albicans in the postgenomic era

M I N I R E V I E W

Identifying infection-associatedgenesofCandidaalbicans in thepostgenomiceraDuncan Wilson1, Sascha Thewes2, Katherina Zakikhany3, Chantal Fradin4, Antje Albrecht1,Ricardo Almeida1, Sascha Brunke1, Katharina Grosse1, Ronny Martin1, Francois Mayer1, Ines Leonhardt1,Lydia Schild1, Katja Seider1, Melanie Skibbe1, Silvia Slesiona1, Betty Waechtler1, Ilse Jacobsen1 &Bernhard Hube1,5

1Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knoell-Institute, Jena,

Germany; 2Institute for Biology, Free University, Berlin, Germany; 3Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm,

Sweden; 4Laboratory of Fundamental and Applied Mycology, University of Lille, Lille, France; and 5Friedrich Schiller University, Jena, Germany

Correspondence: Bernhard Hube,

Department of Microbial Pathogenicity

Mechanisms, Leibniz Institute for Natural

Product Research and Infection Biology, Hans

Knoell Institute (HKI), Beutenbergstrasse 11a,

07745 Jena, Germany. Tel.: 149 3641 532

1401; fax: 149 3641 532 0810;

e-mail: [email protected]

Received 3 March 2009; revised 9 April 2009;

accepted 10 April 2009.

Final version published online 18 May 2009.

DOI:10.1111/j.1567-1364.2009.00524.x

Editor: Teun Boekhout

Keywords

Host-pathogen interactions; invasion; gene

expression; infection models.

Abstract

The human pathogenic yeast Candida albicans can cause an unusually broad range

of infections reflecting a remarkable potential to adapt to various microniches

within the human host. The exceptional adaptability of C. albicans is mediated by

rapid alterations in gene expression in response to various environmental stimuli

and this transcriptional flexibility can be monitored with tools such as micro-

arrays. Using such technology it is possible to (1) capture a genome-wide portrait

of the transcriptome that mirrors the environmental conditions, (2) identify

known genes, signalling pathways and transcription factors involved in pathogen-

esis, (3) identify new patterns of gene expression and (4) identify previously

uncharacterized genes that may be associated with infection. In this review, we

describe the molecular dissection of three distinct stages of infections, covering

both superficial and invasive disease, using in vitro, ex vivo and in vivo infection

models and microarrays.

Introduction

The polymorphic yeast Candida albicans is the most im-

portant fungal pathogen of humans. However, as well as

being a successful pathogen, C. albicans exists as part of the

normal human microbiota and is not found in environ-

mental reservoirs such as soil. In the context of the rest

of the fungal kingdom, this lifestyle is remarkable: of the

estimated 1.5 million (over 100 000 of which have been

confirmed) fungal species, only a very small percentage

(150–200 species) is capable of causing infections in humans.

Some of these species are specialized for infections of the skin

(dermatophytes or Malassezia species), while others are also

able to cause more serious, systemic infections. Most of the

major pathogens in the latter group (such as Aspergillus

fumigatus, Cryptococcus neoformans and Histoplasma capsula-

tum) are environmental fungi, capable of exogenously infecting

susceptible individuals. Candida albicans on the other hand is,

under normal circumstances, a benign colonizer of human

mucosal surfaces and, therefore, highly specialized for life on or

within the human host. However, certain alterations to the host

environment can result in the transition from a commensal to a

pathogenic phase, permitting infection of virtually every organ

of the human body and resulting in severe infections. Even a

mildly compromised immune system or a minor imbalance of

the microbiota can be sufficient for C. albicans to cause

infections of the skin or of mucosal surfaces. These superficial

infections are extremely common – for example, c. 75% of all

women experience vulvovaginal candidosis during their life-

time and a significant proportion suffer from recurrent infec-

tions. In addition, oral and oesophageal candidosis are

particularly common in HIV-positive individuals and, without

intervention with highly active antiretroviral therapy, occurred

in up to 90% of HIV patients (Calderone, 2002). The severity of

candidosis, however, increases dramatically in patient popula-

tions with predisposing factors such as severely impaired

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immunity (e.g. neutropenia), cancer (e.g. leukaemia), disrup-

tion of natural barriers (e.g. by burn injury or disruption of gut

mucosal barriers by abdominal surgery), the presence of

indwelling catheters, dialysis or solid organ transplantation

(Ruhnke & Maschmeyer, 2002; Perlroth et al., 2007). These

mostly hospital-acquired (nosocomial) bloodstream and inva-

sive infections are life-threatening diseases and candidaemia is

now the third most common form of nosocomial bloodstream

infection responsible for 9% of such infections (Wisplinghoff

et al., 2004; Perlroth et al., 2007). Not only are invasive Candida

infections common in intensive care units of hospitals, they are

also difficult to treat (Perlroth et al., 2007). Even with first-line

antifungal therapy, disseminated candidosis has an attributable

mortality of up to 50% in some patient populations, depending

on the underlying illness (Perlroth et al., 2007). The mortality of

severe sepsis caused by Candida is 4 50%, and is therefore

higher than the mortality from sepsis due to any bacterium,

including Pseudomonas aeruginosa. Invasive candidosis is usual-

ly due to fungal entry into the blood resulting in candidaemia

or haematogenous dissemination to internal organs. Candida

albicans can also cause candiduria, perotinitis, endocarditis,

pericarditis, endophthlamitis, meningitis and pneumonia (Cal-

derone, 2002). It is this extraordinary ability of C. albicans to

successfully infect virtually every anatomical site of the human

host that makes it such an important organism to study – both

from a medical and biological perspective.

The exceptional adaptability of C. albicans is mediated by

rapid alterations in gene expression in response to environ-

mental stimuli, such as changes in nutrient availability, pH,

osmolarity, temperature or attack by cells of the immune

system (Fradin et al., 2003; Prigneau et al., 2003; Rubin-

Bejerano et al., 2003; Bensen et al., 2004; Hube, 2004; Brown

et al., 2007; Kumamoto, 2008) and this transcriptional

flexibility can be monitored with tools such as microarrays.

Indeed, this decade has been witness to numerous genome-

wide studies on C. albicans gene expression. These, mainly

in vitro studies have described the transcriptional response

of C. albicans to different environmental conditions such as

the presence of inducers of hyphal development, various

stresses or treatment with antifungal agents (Nantel et al.,

2002; Enjalbert et al., 2006). They have also been essential in

the identification of targets of regulatory circuits (e.g. the

regulon repressed by Tup1 – Garcia-Sanchez et al., 2005).

More recently, we and other groups have used microarray

technology in the context of C. albicans infection biology to (1)

capture a genome-wide portrait of the transcriptome that

mirrors the environmental conditions (thus enabling us to

examine the pathogenic processes in detail), (2) identify

known genes, signalling pathways and transcription factors

involved in pathogenesis, (3) identify new patterns of gene

expression and (4) identify previously uncharacterized genes

(unknown function genes) that may be associated with infec-

tion. As described above, the range of tissue types that

C. albicans can infect is extensive; however, generally infections

can be grouped into two major types: mucosal or systemic

candidosis (also referred to as superficial or invasive infections,

respectively). In our laboratory, we use a cyclical approach for

dissection of the mechanisms of host–pathogen interactions

during distinct types of C. albicans infections and identifying

genes involved in the infection process. First, infection models

are established and investigated using a combination of micro-

scopic and biochemical techniques to determine the temporal

phases of infection. Based on this, appropriate time points are

selected and global transcriptional profiling performed. From

the expression data, genes of interest are selected, deleted and

the resultant knockout mutants tested for attenuation in the

infection model in question. If attenuated, further in-depth

analysis of gene functions will be performed. In this review, we

describe the molecular dissection of three distinct types or

stages of infections covering both superficial and invasive

disease: oral candidosis (mucosal), bloodstream and liver

infection (systemic/invasive).

Mucosal infections: oral candidosis

Recently, we have focused on characterizing oral candidosis

and the molecular mechanisms underpinning this type of

infection using a combination of different in vitro infection

models [e.g. the reconstituted human oral epithelium (RHE)

and monolayers of oral epithelial cells] and by comparing

these data with results generated from clinical samples (mainly

from HIV-positive patients suffering from oral candidosis).

Based on our observations made in the experimental infection

models, we identified three different substages during the

pathogenesis of oral infections: an early/colonization phase,

characterized by adhesion of the fungus to the upper layers of

the host tissue and fungal proliferation; an invasion phase,

associated with hyphal formation and penetration of the

upper cell layers of the oral tissue; and a late phase, associated

with substantial tissue destruction (Fig. 1a and b). By combin-

ing histological analysis, scanning electron microscopy, cellu-

lar cytotoxicity measurements and global gene-expression

analysis, we were able to study these different stages of oral

candidosis at both the cellular and molecular level. Of course,

naturally, the time scale of oral candidosis is different. Here,

initial colonization can precede the onset of infection by many

years and it is only upon compromise of the host that oral

candidosis manifests. However, despite the artificiality of the

RHE model, it appears to at least partially mimic the clinical

setting as the majority of genes constitutively expressed in the

RHE were also upregulated by C. albicans infecting the oral

cavity of HIV-positive patients (Zakikhany et al., 2007).

Early-phase oral candidosis

The early phase (0–3 h) in the experimental setting repre-

sents the establishment of infection: inoculated C. albicans

FEMS Yeast Res 9 (2009) 688–700 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

689Identifying infection-associated genes of Candida albicans

yeast cells that come into contact with the epithelium adhere

to host cells and this contact results in the yeast to hypha

transition (Fig. 1a and b). These two events – hyphal

formation and adhesion – were also reflected at the mole-

cular level with a number of known genes encoding adhesins

or other hyphal-associated genes upregulated during the

early phase, including HWP1, FKH1, ATP2, TEF1, ALS3 and

SOD5 (Fig. 1c). Following contact with the epithelium,

hyphal formation allows tight receptor-mediated contact

between fungal and host cells. This interaction in turn

results in reorganization of the host cytoskeleton, envelop-

ment of the fungal cell by membrane-derived pseudopod-

like structures and subsequent uptake of the fungal cell (Fig.

1a and b). Such a microorganism-triggered epithelial-driven

invasion process, known as induced endocytosis, is well

described for bacteria such as Salmonella, Shigella or Yersinia

(Isberg, 1996; Goosney et al., 1999; Tran Van Nhieu et al.,

2000) and has, in the case of C. albicans, recently been

shown to be mediated by binding of Als3 on the surface of

the fungus to oral epithelial cell E-cadherin (Filler &

Sheppard, 2006; Phan et al., 2007). The fact that ALS3

expression is highly induced following C. albicans–epithelial

contact corroborates this finding (Fig. 1c). Therefore, dur-

ing the early attachment phase, C. albicans has already begun

epithelial invasion via induced endocytosis.

This early phase of germ tube formation, attachment and

induced endocytosis is followed by the invasion phase

(3–12 h). The invasion phase is characterized by extensive

epithelial penetration via prolific hyphal growth and the

expression of several hyphal-associated genes. Although this

Fig. 1. The distinct stages of oral Candida albicans infection. (a) The progression of C. albicans infection of human oral epithelial cells, characterized by

attachment, induced endocytosis, active penetration and tissue destruction; (b) representative scanning electron micrographs of the different stages of

infection; note the engulfment of fungal cells by epithelial-derived pseudopod-like structures during induced endocytosis; (c) expression profiles of

selected genes during infection of the human oral RHE. The colours indicate the degree of (fold) expression: o 0.4, green; 0.4–0.7, light blue; 0.7–1.5,

beige; 1.5–2, yellow; 2–3, light orange; 3–5, orange; 4 5, red.

FEMS Yeast Res 9 (2009) 688–700c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

690 D. Wilson et al.

phase is associated with considerable invasion of the epithe-

lium, substantial tissue damage is not observed until the late

phase (12–24 h), suggesting that initial invasion alone is

not sufficient to cause damage and not the only factor

contributing to tissue destruction. Invasion during the mid

and late phases is mediated predominantly by active pene-

tration (Fig. 1a and b). Active penetration is an invasion

mechanism distinct from induced endocytosis as it does not

rely on the host’s cellular machinery, but exclusively on

fungal attributes possibly including physical pressure ap-

plied by the advancing hyphal tip and the secretion of

extracellular hydrolases, which have been presumed to assist

in the invasion process via the degradation of host cell

components (Schaller et al., 2005).

Late-phase oral candidosis

Finally, the late phase, characterized by substantial tissue

destruction, was – at the transcriptional level – associated

with numerous adaptive responses (Fig. 1c). By 24 h,

expression of the two alkaline-responsive genes, PHR1 and

PRA1, was induced, as was that of the alkaline-responsive

transcription factor, RIM101, suggesting that the fungal cells

were in an environment of neutral-alkaline pH. Although it

is possible that the induction of these genes was influenced

by the pH of the RHE maintenance medium, it is probable

that these genes are truly induced during oral epithelial

tissue destruction, as their transcript levels were 4 1.5-fold

upregulated in patient samples. Candida albicans appears to

face a glucose-poor environment in the oral tissue as

indicated by the upregulation of the glucose and maltose

transporter genes HGT12 and MAL31 and key components

of the gluconeogenesis pathway and glyoxylate cycle (PCK1,

MLS1 and ICL1). A number of genes involved in amino acid

sensing and transport (GNP1, CAR1 and CAR2) were also

upregulated suggesting that C. albicans cells sense a nitro-

gen-poor environment on or within the oral tissue. In

addition, strong induction of the high-affinity phosphate

transporter PHO84 suggested that ready access to phosphate

sources was also limited in this tissue. Apart from the

limitation of certain nutrients, the only other clear stress

condition encountered by the fungus appeared to be nitro-

sative stress as indicated by the upregulation of the marker

genes YHB5, SSU1 and YHB1 involved in detoxification of

nitric oxide (Hromatka et al., 2005). It is known that

epithelial cells produce nitrogen monoxide radicals as part

of their innate immune response against microorganisms

and it would therefore appear that the experimental RHE

model is capable of mounting at least this innate defence

mechanism against C. albicans infection. Surprisingly, many

well-known genes involved in iron acquisition – a major

virulence determinant of virtually all pathogenic microor-

ganisms (Andrews et al., 2003; Howard, 2004; Sutak et al.,

2008) – were not upregulated in oral RHE tissue. It is

possible that the experimental set-up of the RHE allowed

exposure of C. albicans to unnaturally high levels of iron

from the surrounding medium. Alternatively, C. albicans

may be utilizing a novel iron source during invasion of this

tissue. Our group has recently demonstrated that C. albicans

can utilize iron from host ferritin during oral infections and

have shown that this event is mediated by the multifunctional

cell surface protein Als3 (Almeida et al., 2008). Given the high

expression of ALS3 during our models of oral infection, this

novel iron-acquisition strategy is a likely possibility.

Molecular analysis of an oral candidosis-associated gene

Among the genes that were upregulated during oral infec-

tion were a substantial number with no known function or

with no homologue in the brewer’s yeast Saccharomyces

cerevisiae. We reasoned that these unknown function infec-

tion-associated genes constitute good candidates for novel

virulence factors in C. albicans. One such gene, orf19.7561

(renamed EED1) has no obvious homologues in any other

sequenced organism and was upregulated in patient samples

and during both the early and the late phases of RHE

infection. Given the expression profile of this gene we

predicted that it might play a role (1) during the onset of

and (2) in the maintenance/persistence of infections of the

oral cavity. For functional characterization of the role of

EED1 during oral infections, both copies of the gene were

deleted. The resultant eed1D mutant had severe hyphal-

formation defects, growing as yeast or short chains of

pseudohyphae under standard laboratory hyphal-induction

conditions. Growth in the presence of very strong stimuli

(e.g. RPMI with 10% serum) resulted in elongated pseudo-

hyphal germ tube formation of eed1D; however, following

extended incubation time (5 h), the eed1D cells were unable

to maintain filamentous growth and switched back to the

yeast morphology. These results suggested that EED1 is

required for both the initiation and maintenance of fila-

mentous growth. As hyphal development is a prerequisite

for invasion of oral epithelial cells, it was predicted that the

eed1D mutant would be highly attenuated in our oral

infection models. Surprisingly, despite the observed in vitro

filamentation defects, upon infection of TR146 oral cells the

eed1D mutant cells switched to filamentous growth, reinfor-

cing the view that fungal contact with epithelial cells itself is

a potent inducer of filamentation and bypasses the require-

ment for EED1. Moreover, these filaments were able to

invade the epithelial cells via induced endocytosis. Despite

initial filamentation and invasion, by 24 h, eed1D had

reverted to the yeast morphology and existed within intrae-

pithelial inclusion bodies while wild-type cells disseminated

throughout the epithelial tissue via extensive hyphal

FEMS Yeast Res 9 (2009) 688–700 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

691Identifying infection-associated genes of Candida albicans

formation. The gene was therefore named EED1 for epithe-

lial escape and dissemination. Although other genes (EFG1

and CPH1) have been shown to be required for escape from

professional phagocytic cells, such as macrophages, EED1 is

the first example of a fungal gene required for escape from

and dissemination within oral epithelial tissue.

Systemic infections: survival in the bloodand liver invasion

Systemic infections are characterized by three major events:

dissemination via the bloodstream followed by escape from

the bloodstream and the infection of deep-seated organs. In

order to study these events, we have established bloodstream

and liver infection models. A number of mechanisms as to

how C. albicans enters the bloodstream have been proposed;

these include so-called ‘natural’ routes, such as via the

penetration of epithelial cells at mucosal surfaces, or iatro-

genic (artificial) routes, such as implantation of medical

devices, surgery, trauma or depletion of the natural micro-

bial flora by antibiotic treatment (Mavor et al., 2005). Once

inside the bloodstream, C. albicans is able to disseminate

and can potentially infect almost every organ of the host;

however, for this to occur, the fungus must first survive

within the bloodstream and then escape via traversal of the

endothelium.

Survival in blood

The bloodstream is a harsh environment for any pathogenic

microorganism due to the presence of numerous immu-

noactive cells and molecules; however, in order to cause

systemic infections, pathogenic microorganisms must pos-

sess mechanisms to resist attack by the immune system. In

order to investigate the fungal response to the hostile

environment of the blood, we incubated C. albicans with

human blood and measured the transcriptional response of

the fungus over a time course experiment (10, 20, 30 and

60 min) using microarrays compromising 2002 genes (Fra-

din et al., 2003). In this study, we showed that C. albicans

rapidly adapts to the blood environment. Such rapid adap-

tation relies on the expression of a distinct subset of genes

and translation into the corresponding proteins necessary to

meet the requirements of the new environment. This was

reflected by the strong upregulation of genes related to

protein synthesis at 10 min. This early adaptation event was

followed by the upregulation of genes involved in the

glyoxylate cycle, fermentation, glycolysis and response to

oxidative stress. In addition, several known hyphal-asso-

ciated genes were upregulated upon exposure to blood, in

agreement with microscopy observations that 42% of cells

had undergone the yeast to hypha transition during the time

course of the experiment (Fig. 2a). The fact that genes of the

glyoxylate cycle were upregulated at the same time as genes

involved in glycolysis is surprising: in general, the glycolytic

pathway and the glyoxylate cycle are utilized in the absence

or presence of carbohydrates, respectively, and not at the

same time by the same cell. One explanation for this is that

the fungal cells existed in at least two distinct subpopula-

tions, one with and one without ready access to sugars. This

split-population hypothesis may also be supported by the

morphogenic heterogeneity of fungal cells exposed to blood

and may be explained by the fact that blood consists of a

heterogeneous mixture of different cell types, which may act

differentially on distinct fungal cells.

In order to dissect which blood factors are involved in

combating C. albicans, and which fungal factors may resist

this assault, we further analysed the cellular and transcrip-

tional response of C. albicans to whole blood and to various

blood fractions enriched in particular host cell types (Fradin

et al., 2005). One major virulence trait – the yeast to hypha

transition – is also presumed to aid in C. albicans escape

from the bloodstream by assisting in traversal of the

endothelial lining of blood vessels (Filler & Sheppard,

2006) and has been shown to mediate escape following

phagocytosis by macrophages (Lorenz & Fink, 2001). The

morphology of C. albicans exposed to the various blood

fractions for 30 min was therefore determined. In blood

fractions lacking neutrophils (erythrocyte, monocyte/

lymphocyte and plasma fractions) most cells (80–85%)

formed germ tubes. However, exposure of C. albicans to the

polymorphonuclear (PMN) neutrophil fraction almost

completely blocked hyphal development with 96.5% of cells

remaining in the yeast morphology (Fig. 2a). This dominant

effect of PMNs on the morphology of C. albicans was also

reflected at the transcriptional level: cluster analysis showed

that cells incubated in whole blood or in the PMN fraction

shared a similar profile that was distinct from cells incubated

in the presence of plasma, erythrocytes, monocytes or in

whole blood depleted of PMNs.

In the absence of neutrophils, numerous genes associated

with protein synthesis, glycolysis and hyphal formation

were expressed, suggesting that cells were metabolically

active and in concordance with observation that the

majority of cells had undergone the yeast to hypha transi-

tion. In stark contrast, in the presence of neutrophils, fungal

cells underwent growth arrest, and hyphal morphogenesis

was almost completely blocked. Nutrient starvation cer-

tainly appeared to contribute to the observed growth arrest.

The environmental nitrogen level of cells exposed to neu-

trophils was low as indicated by the upregulation of the

ammonium permeases MEP2 and MEP3. Furthermore, the

amino acid starvation-responsive transcriptional regulator

GCN4, as well as several genes associated with arginine,

leucine, lysine and methionine biosynthetic pathways were

induced as described previously by Rubin-Bejerano et al.

(2003). Vacuolar proteases (Prb1, Prb2 and Apr1) and

FEMS Yeast Res 9 (2009) 688–700c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

692 D. Wilson et al.

carboxypeptidases (Prc1 and Prc2) – known to be involved

in the utilization of endogenous nitrogen sources – were also

upregulated in response to neutrophils. In addition to this

clear nitrogen-starvation response, C. albicans also appeared

to face a carbohydrate-poor environment, as genes associated

with glycolysis were downregulated while components of the

glyoxylate cycle were strongly induced (Fig. 2c).

One of the proposed antimicrobial mechanisms of neutro-

phils is the production of reactive oxygen species, which

contribute to the killing of pathogenic microorganisms by

attacking multiple cellular components such as DNA, proteins

and lipids. It appears that neutrophils exert substantial

oxidative stress on C. albicans as a number of genes involved

in detoxification of reactive oxygen species – such as super-

oxide dismutases, catalase, glutathione peroxidase, glutathione

reductase, glutathione S-transferase and thioredoxin – were

strongly induced in response to incubation with neutrophils

(Fig. 2c). To further investigate the role of oxidative stress on

C. albicans during interactions with neutrophils we fused a

green fluorescent protein (GFP) reporter to the oxidative

stress-responsive SOD5 promoter (Martchenko et al., 2004).

The SOD5 reporter was found to be induced in yeast cells that

were either attached to or phagocytosed by neutrophils. This

finding suggests that neutrophils elicit an oxidative stress

response, which does not rely on phagocytosis of the fungal

cell. Finally, it was shown that sod5D mutant cells had reduced

survival following incubation with neutrophils, reinforcing the

view that an appropriate oxidative stress response is critical for

Fig. 2. Interaction of Candida albicans with blood. (a) The effect of blood or blood fractions on C. albicans morphology, note the heterogenous

morphology of fungal cells in whole blood, repression of hyphal formation in the PMN (neutrophil) fraction and hyphal development in the MNC

(monocyte/lymphocyte) and red blood cells (RBC) (erythrocyte) fractions; (b) representative micrographs of human blood cells; (c) expression profiles of

selected genes following 30-min incubation with indicated blood component. The colours indicate the degree of (fold) expression: o 0.4, green;

0.4–0.7, light blue; 0.7–1.5, beige; 1.5–2, yellow; 2–3, light orange; 3–5, orange.

FEMS Yeast Res 9 (2009) 688–700 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

693Identifying infection-associated genes of Candida albicans

fungal survival in the hostile environment of the blood and

subsequent dissemination throughout the body.

Liver invasion

Having survived the bloodstream for a certain time period,

C. albicans cells must escape from the circulation via

traversal across the endothelial lining of blood vessels in

order to avoid the killing activities of blood cells. Following

escape from the blood stream, the fungus has the potential

to infect virtually every internal organ. As a model of

infection of a deep-seated organ we analysed C. albicans

invasion of the liver. In order to identify genes associated

with tissue invasion of organs, we designed two comparative

experiments (Thewes et al., 2007a, b): (1) comparison of

C. albicans gene expression following in vivo intraperitoneal

infection of mouse liver and ex vivo infection of perfused pig

liver to identify genes commonly associated with infection

of this organ; (2) analysis of genes expressed in the liver

model by the invasive strain (SC5314) but not by the

noninvasive strain (ATCC10231) to more stringently define

those genes actually involved in the invasion process, as

opposed to those that are simply more highly expressed in

the host than in broth culture. Preliminary histological

Fig. 3. Candida albicans invasion of liver tissue. (a) Candida albicans infection of in vivo mouse or ex vivo pig liver, characterized by initial attachment

and hyphal formation, superficial invasion of the liver capsule followed by extensive invasion into deeper liver tissue; (b) representative histological

micrographs of C. albicans liver infection; (c) expression profiles of selected genes during infection of the mouse liver at the time of attachment (0.5 h),

superficial (3 h) and deeper (5 h) invasion. The colours indicate the degree of (fold) expression: 0.4–0.7, light blue; 0.7–1.5, beige; 1.5–2, yellow; 2–3,

light orange; 3–5, orange; 4 5, red.

FEMS Yeast Res 9 (2009) 688–700c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

694 D. Wilson et al.

analysis revealed that in both infection models, the infection

process of the invasive strain was characterized by initial

attachment and hyphal formation followed by penetration

of the liver capsule and invasion into the tissue by hyphal

cells (Thewes et al., 2007b; Fig. 3a and b).

Based on the expression profile of so-called ‘marker

genes’, a number of inferences could be drawn about

the nutritional state of the environment encountered by

C. albicans during liver invasion (Thewes et al., 2007a;

Fig. 3c). For example, as opposed to the situation in the oral

cavity, there appeared to be sufficient access to sugar for

utilization as a carbon source as indicated by upregulation

of PFK1 (encoding a key enzyme of glycolysis), PDA1 and

PDX1 (involved in acetyl-CoA biosynthesis) and KGD1 and

KGD2 (encoding key enzymes of the tricarboxylic acid

cycle). Similarly, there appeared to be sufficient levels of

nitrogen in the liver as genes associated with amino acid

starvation were not upregulated. In spite of this apparent

abundance of carbon and nitrogen sources, C. albicans

appeared to face severe iron limitation upon liver invasion

as indicated by the upregulation of FET5, FTR1, ZRT1,

CFL1, RBT5, FRE5 and CTR1 – all genes associated with an

iron-poor environment or with iron acquisition. There did

not appear to be any specific response to oxidative, nitrosa-

tive or osmotic stress in the liver, although genes associated

with general/thermal stress such as HSP78, HSP90, HSP104,

HSP12 and HSP70 were upregulated. Whether this is due to

actual thermal stress or cross-protection against an as-yet

unknown stress in the liver remains to be determined.

Upregulation of well-known hyphae-associated genes such

as SAP5, ALS3 and HWP1 was in accordance with the

histological observation that cells were growing predomi-

nantly in the hyphal morphology. Finally, upregulation of

the alkaline-responsive PHR1 indicated that the majority of

cells encountered a neutral-alkaline pH.

Molecular analysis of a gene involved in invasivecandidosis

The next step of this study involved dissection of the

transcriptional data to identify genes intimately associated

with the process of liver tissue invasion. To focus our analysis

in this direction, we looked for genes that were upregulated

by the invasive SC5314 during both mouse (3 and 5 h

postinfection) and pig (12 h postinfection) liver invasion but

not by the noninvasive strain, ATCC10231. This detailed

analysis yielded five genes, one of which (DFG16) was selected

for further analysis. DFG16 encodes a member of the PalH/

RIM21 super-family, which constitutes putative pH sensors

and has been shown to function in the Rim101 pathway

(Barwell et al., 2005). To characterize the function of this

gene, both alleles were deleted and the resultant dfg16Dmutant tested. In accordance with the protein’s predicted role

as a pH sensor, dfg16D behaved normally at acidic pH but was

unable to grow under iron- or phosphate-limited conditions

at alkaline pH. Moreover, dfg16D had reduced osmotic stress

tolerance at alkaline, but not acidic, pH and failed to form

filaments at pH 8. Finally, the virulence of dfg16D was

attenuated in a mouse model of haematogenously dissemi-

nated candidosis (Thewes et al., 2007a) and dfg16D cells had

reduced potential to invade liver tissue following intraperito-

neal infection (our unpublished data).

The fact that transcript levels of DFG16, encoding a

putative pH sensor, are increased during liver invasion

underscores the importance of environmental pH sensing

and adaptation during the progression of systemic candido-

sis. Unlike environmental human fungal pathogens, which

receive clear host-associated signals to initiate infection (e.g.

a shift to 37 1C), C. albicans must be able to dramatically

reprogramme its behaviour based on more subtle environ-

mental cues. Probably the most extensively studied trait of

C. albicans, the yeast to hypha transition, is under the

control of an extensive network of signalling pathways,

which integrate the receipt of a wide range of environmental

signals (temperature, pH, oxygen, CO2, nutrients and physical

contact to name only a few) to control cellular morphology.

Why must C. albicans sense such a vast array of environmental

signals to determine its morphology? Firstly, unlike many

environmental pathogenic fungi, which form either non-

pathogenic/saprophytic or host-associated/pathogenic mor-

phologies, the pathogenicity of C. albicans relies on the

reversible yeast to hypha transition (mutants unable to form

yeast also have reduced virulence). Secondly, because

C. albicans is continuously in contact with the host (even in

the nonpathogenic stage as commensal) and, therefore, in an

environment of physiological temperature, the formation of a

single morphological state in response to temperature alone

would be inappropriate. Thirdly, although the gross morphol-

ogy of C. albicans is dependent on certain, sometimes quite

different, combinations of environmental signals, any given

subset of signals may specifically result in the expression of a

different subset of genes not strictly coexpressed with a given

morphology; these discrete transcriptomes reflecting the given

requirements encountered at particular anatomical niches: for

example, RBT5, encoding the cell surface-localized haemoglo-

bin-binding protein (Weissman & Kornitzer, 2004), is highly

expressed by hyphal cells invading liver tissue but not by

hyphal cells invading oral tissue.

Given the highly dynamic response of C. albicans to its

environment, combined with the diverse niches it occupies, it

is not surprising that the genome of this fungus contains a

large number of genes encoding described and putative

sensors. The correct sensing of – and response to – environ-

mental signals is crucial and relies on the expression of

relevant sensor-encoding genes in a given microenvironment.

The reduced virulence of dfg16D demonstrates that

FEMS Yeast Res 9 (2009) 688–700 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

695Identifying infection-associated genes of Candida albicans

perturbations in the sensing of a single environmental factor

(pH) can block the pathogenicity of C. albicans and illustrates

the delicacy of the cross-talk between the host and the

pathogen.

Discussion and outlook: the future ofC. albicans infection models andidentification of infection-associatedgenes

In summary, we have presented three examples of how,

using carefully designed infection models combined with

global gene expression analysis, we can confirm presumed

features of pathogenicity, identify genes that are expressed

during infection and discover novel aspects of host–patho-

gen interactions during representative C. albicans infections.

The transcriptome of C. albicans as a tool toexplore the physiological environment duringinfection

One possible application of genome-wide profiling of a

pathogenic microorganism during infection is the concept

of the transcriptome as a biosensor, which may enable us

to monitor the physiological conditions encountered by

the microorganism. For example, we verified that when

C. albicans was exposed to blood, neutrophils elicit a strong

oxidative stress response. This was not the case in the liver or

oral cavity, where general or nitrosative stress responses,

respectively, dominated, thus demonstrating that C. albicans

faces diverse challenges depending on the anatomical niche

in which it finds itself. On the other hand, some features of

the host environment appeared to be common and there

was significant transcriptional overlap between different

infection types. For example, key components of the glyox-

ylate cycle were upregulated during both oral and blood-

stream (but not liver) infections. Similarly numerous

hyphal-associated genes were upregulated in both oral and

liver infections, but not during interaction with neutrophils.

Interestingly, very few genes were strongly induced under all

infection conditions, suggesting that, in general, there is no

general response to infection-associated growth and that the

transcriptome of C. albicans within the human host is

overall niche specific. This may be explained by the com-

mensal lifestyle of C. albicans. Environmental fungi receive

clear signals upon the onset of infection. For example,

synthesis of one of the major virulence factors of C. neofor-

mans, the polysaccharide capsule, is induced upon infection

via the fungus sensing a shift to an environment of iron

limitation and physiological temperature (Jung & Kronstad,

2008). Candida albicans on the other hand, outside of the

laboratory, is in constant contact with the human host and

while colonizing mucosal surfaces is constitutively exposed

to an environment of physiological temperature and limited

iron. Only disturbance of the normal bacterial microbiota

and/or a weakened immune system triggers activity asso-

ciated with infection and the colonization of new niches

within the human body. Given the niche specificity of

C. albicans, the principle of using the transcriptome as a

biosensor may be applicable for studying the conditions

faced by the fungus at diverse body sites. For example, based

on detailed in vitro studies, the expression of certain marker

genes such as PHR1, PRA1, PHR2, SOD5, CAT1, YHB1,

ICL1 and MEP2 may indicate alkaline or acidic pH, oxida-

tive stress, nitrosative stress, low glucose or nitrogen condi-

tions, respectively (De Bernardis et al., 1998; Sentandreu

et al., 1998; Lorenz & Fink, 2001; Martchenko et al., 2004;

Biswas & Morschhauser, 2005; Corvey et al., 2005; Hromat-

ka et al., 2005). However, further in vitro gene expression

experiments are needed to identify not only single marker

genes, but also sets of genes (regulons) and signatures

associated with a certain physiological situation. For exam-

ple, the identification of particular transcriptional signa-

tures associated with growth on defined nutrient sources

could be used to overlay the transcriptome during infection

and may actually pinpoint what nutrients are being utilized

in vivo. Bignell and colleagues (McDonagh et al., 2008) have

recently used such an approach to study the initiation of

infection during pulmonary aspergillosis. By comparing

transcriptional signatures obtained from specific in vitro

conditions to the expression pattern observed upon infec-

tion of the murine lung, they demonstrate that A. fumigatus

adaptation to the host is associated with iron limitation,

alkaline stress and nutrient deprivation.

Unknown function genes

One major obstacle in interpreting such global studies is the

overall lack of annotation of a large set of genes in the

C. albicans genome. Although d’Enfert et al. (2005) assigned

‘tentative functional assignments’ for 92% of the C. albicans

genome, the true number of genes with known function is

probably much lower. Braun et al. (2005) reported gene

ontology (GO) terms (excluding unknown function) for only

56% of the genome. Moreover, 19% of genes do not share

significant sequence homology with other organisms (Braun

et al., 2005). Because C. albicans is highly specialized to exist

in association with warm-blooded mammals, it is reasonable

to hypothesize that C. albicans possesses unique factors

involved in interactions with its host environment. Although

these host-interaction factors likely evolved to maintain a

commensal life-style, when the host environment undergoes

certain changes (antibiotic treatment, immune compromise,

etc.), they may become virulence factors involved in the onset

and progression of invasive infections. Therefore, one of our

central premises is that unknown function genes

FEMS Yeast Res 9 (2009) 688–700c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

696 D. Wilson et al.

transcriptionally upregulated during infection constitute pro-

mising host-interaction/virulence factors, required for the

pathogenesis of C. albicans. For example, we have shown that

EED1, a previously undescribed gene with no obvious

sequence homology in any other sequenced organism is

induced during in vivo and in vitro oral infections and was

essential for dissemination within oral tissue (Zakikhany

et al., 2007). This example therefore illustrates that there exist

genes of unknown function, induced during infection, which

are essential for the pathogenesis of C. albicans. We are

therefore continuing to explore the role of unknown function

genes in the pathobiology of C. albicans.

Experimental design

A major issue to consider during the interpretation of

current transcriptional profiling studies is the choice of

control. In our group we cohybridize experimental RNA

against a ‘common control’ of RNA from cells grown in YPD

at 37 1C to mid-logarithmic phase. The rationale behind this

is that virtually all genes are expressed under this condition,

and many infection-associated genes should be more highly

expressed during infection than in batch culture. However,

care should be taken when using this approach as ‘upregula-

tion’ is dependent on the relative expression in the control

and thus genes expressed at the same or even lower level

in vivo compared with YPD may still be important for the

infection process.

Recent work by Walker et al. (2009) analysing gene

expression of C. albicans infecting the rabbit kidney further

highlighted certain issues associated with the choice of

control. The authors report a strong downregulation in the

expression of well characterized and reportedly ‘hyphae-

associated’ genes such as ALS3 and ECE1, despite histologi-

cal observations suggesting hyphal growth in the kidney

lesions. However, in this study the control chosen was RNA

from cells grown in RPMI 1640 at 37 1C and, as the authors

discuss, the downregulation of these genes does not neces-

sarily mean that they are not being transcribed in vivo, but

simply that the expression is significantly higher in the

control cells.

Two strategies can be used to circumvent these issues. The

first is to design a temporal experiment where the transcrip-

tome is captured at different time points during the infec-

tion process including the preculture. The dynamics of

expression can then be analysed over time, effectively

independent of the control signal. The second approach is

to perform parallel experiments where only one variable

is changed between the two, for example, the comparison of

C. albicans infecting reconstituted human epithelium either

with or without the presence of neutrophils (Schaller et al.,

2004). It should be noted that, for some experimental

designs, one colour labelling, may be more appropriate.

Readers are directed to two excellent reviews describing the

design principles of microarray experiments (Yang & Speed,

2002; Bryant et al., 2004).

Although microarrays represent the current ‘gold stan-

dard’ for unbiased global analysis of gene expression in

C. albicans, other technologies may be more appropriate for

certain applications. For example, a number of reporter

systems have been described that can monitor expression of

single genes during models of infection. For example, in vivo

expression can be measured very sensitively by placing the

site-specific recombinase FLP under control of the promoter

of interest. Using this method, transient expression of

the gene of interest can be detected during animal models

of infection at the single cell level (Staib et al., 1999). A

second single cell profiling approach involves fusion of the

promoter of interest to a GFP reporter. This method is

particularly effective for monitoring gene expression in a

mixed population of cells as demonstrated by Barelle et al.

(2006).

Finally, although the C. albicans genome now stands at its

21st assembly (van het Hoog et al., 2007), it is likely that

numerous small transcripts, potentially involved in patho-

genesis, have been overlooked and, therefore, not included

on current microarrays. This and the above-discussed

problems may be overcome by serial analysis of gene

expression of C. albicans during infection as has recently

been described for C. neoformans infecting the murine lung

(Hu et al., 2008). Using this approach, it will be possible to

determine which genes are actually expressed at high levels

during infection and to uncover truly novel transcripts

involved in the infection process.

The future of C. albicans infection models

The past half decade has witnessed the establishment of

numerous postgenomic technologies and refined infection

models for the study of C. albicans, and this review has

described the use of such technologies to characterize the

behaviour of this fungus in different niches of the human

host. However, this is only part of the story, as all forms of

candidosis (whether superficial or invasive infections) are

dependent on the host status and the host response must

always be considered as an element of pathogenesis

(Richardson & Rautemaa, 2009). Therefore, we need to also

investigate the specific host responses for the different types

of infection and the different stages of disease development

to understand the underlying mechanisms that contribute

to C. albicans infections.

Based on infection models such as those presented in this

review, it should be possible to extend current studies on

C. albicans host–pathogen interactions to more comprehen-

sively cover infection-associated parameters: both fungal

pathogenicity mechanisms and host immune factors. For

FEMS Yeast Res 9 (2009) 688–700 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

697Identifying infection-associated genes of Candida albicans

example, we have shown that in blood, it is neutrophils that

have the greatest impact on C. albicans morphology, viabi-

lity and gene expression (Fradin et al., 2003, 2005) lending

direct experimental evidence to clinical observations that

neutrophils are the primary defence mechanism against

systemic candidosis (Chauhan et al., 2006). As an example

of extension of such a study, Fradin et al. (2007) went on to

characterize the transcriptional response of neutrophils

exposed to C. albicans, identifying an enrichment in genes

involved in proinflammatory cell–cell signalling. And while

the innate immune system is vital for the first line of

protection against systemic candidosis, T-cell-mediated cel-

lular immunity is generally considered particularly impor-

tant at oral mucosa (reviewed in Saunus et al., 2008).

Recently, Schaller and colleagues (Schaller et al., 2004;

Weindl et al., 2007) have utilized the RHE model to show that

neutrophils induced a protective T-helper type 1 immune

response in human oral epithelial cells and that this protection

is directly mediated by TLR4 receptors.

The future of C. albicans infection biology will surely

extend such approaches as those reviewed here, simulta-

neously integrating analysis of pathogen and host factors to

assemble both a detailed and global picture of host–patho-

gen interactions. The inherent complexity of these interac-

tions means that systems biology must also play a role in

shaping our understanding of this delicate cross-talk.

Attaining such an inclusive portrait of candidosis will un-

questionably drive forward the discovery of novel diagnostic

tools and the development of effective antifungal therapies.

Acknowledgements

Our own research was supported by the Robert Koch-Institute

(RKI), the Hans-Knoell-Institute (HKI), the Deutsche

Forschungsgemeinschaft (DFG), the European Union (EU),

and the Federal Ministry of Education and Research (BMBF).

We would also like to thank the reviewers of this manuscript

for their helpful suggestions and Brice Enjalbert for interesting

discussions on data interpretation.

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