some biological features of candida albicans mutants for genes coding fungal proteins containing the...

R E S E A R C H A R T I C L E

Somebiological featuresofCandidaalbicansmutants forgenescoding fungal proteins containing theCFEMdomainAna Perez1, Gordon Ramage2, Rosario Blanes1, Amelia Murgui1, Manuel Casanova1 & Jose P. Martınez1

1Departamento de Microbiologıa y Ecologıa, Facultad de Farmacia, Universitat de Valencia, Burjasot, Valencia, Spain; and 2Infection and Immunity

Research Group, Glasgow Dental School, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK

Correspondence: Jose P. Martınez,

Departamento de Microbiologıa y Ecologıa,

Facultad de Farmacia, Universitat de Valencia,

46100 Burjasot, Valencia, Spain. Tel.: 134

963 544 770; fax: 134 963 544 544;

e-mail: [email protected]

Received 17 July 2010; revised 4 December

2010; accepted 12 December 2010.

Final version published online 17 January 2011.

DOI:10.1111/j.1567-1364.2010.00714.x

Editor: Richard Calderone

Keywords

Fungal cell surface proteins; PGA10, RBT5 and

CSA1/WAP1 genes; biofilms; atomic force

microscopy; cell surface hydrophobicity.

Abstract

Several biological features of Candida albicans genes (PGA10, RBT5 and CSA1)

coding for putative polypeptide species belonging to a subset of fungal proteins

containing an eight-cysteine domain referred as common in several fungal

extracellular membrane (CFEM) are described. The deletion of these genes

resulted in a cascade of pleiotropic effects. Thus, mutant strains exhibited higher

cell surface hydrophobicity levels and an increased ability to bind to inert or

biological substrates. Confocal scanning laser microscopy using concanavalin A-

Alexafluor 488 (which binds to mannose and glucose residues) and FUN-1 (a

cytoplasmic fluorescent probe for cell viability) dyes showed that mutant strains

formed thinner and more fragile biofilms. These apparently contained lower

quantities of extracellular matrix material and less metabolically active cells than

their parental strain counterpart, although the relative percentage of mycelial

forms was similar in all cases. The cell surface of C. albicans strains harbouring

deletions for genes coding CFEM-domain proteins appeared to be severely altered

according to atomic force microscopy observations. Assessment of the relative

gene expression within individual C. albicans cells revealed that CFEM-coding

genes were upregulated in mycelium, although these genes were shown not to

affect virulence in animal models. Overall, this study has demonstrated that CFEM

domain protein-encoding genes are pleiotropic, influencing cell surface character-

istics and biofilm formation.

Introduction

Candida albicans is unique among fungal pathogens in

terms of the diversity of infections it can cause. The fungus

is a normal commensal on the mucosal surfaces of the

gastrointestinal and urogenital tract without clinical symp-

toms in the majority of humans, but by contrast to numerous

other commensals, C. albicans has the ability to colonize and

invade host tissues when the host immune system is weak or

when the competing flora is eliminated, usually causing

superficial infection of mucosal epithelium (Calderone,

2002). However, in immunocompromised individuals, infec-

tions can progress to a severe systemic invasion, leading to a

life-threatening situation. In this context, C. albicans is the

major fungal pathogen in humans (Calderone, 2002), and

recent surveys in the United States have shown that Candida

species are the third to fourth most commonly isolated

bloodstream pathogen, having surpassed gram-negative rods

in frequency as causative agents of septicaemia in humans

(Edmond et al., 1999; Diekema et al., 2002).

The virulence factors expressed or required by C. albicans to

invade the host may vary depending on the type of clinical

manifestation (i.e. mucosal or systemic), the site and stage of

infection, and the nature of the host response. It seems apparent

that virulence in C. albicans is multifactorial and although many

virulence traits have been suggested, the production of extra-

cellular hydrolytic enzymes, hyphae formation (morphologic

transition or dimorphism), phenotypic switching, the presence

of surface recognition molecules (adhesins and receptors for

ligands in host tissues) and the ability to form biofilms have

been the most widely studied (Calderone & Fonzi, 2001; Soll,

2002; Sudbery et al., 2004; Hube, 2006; Ramage et al., 2009).

Adherence of C. albicans to host cells is seen as an essential

early step in the establishment of disease. Attachment of

FEMS Yeast Res 11 (2011) 273–284 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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microorganisms to tissues is a complex process, involving

both specific receptor molecules and nonspecific physical and

chemical cell surface properties. The most important factors

mediating adhesiveness are considered to be hyphal morpho-

genesis, cell surface hydrophobicity (CSH) and the presence of

cell surface adhesins and receptors for host ligands (Hazen &

Glee, 1995; Sundstrom, 1999, 2002; Calderone et al., 2000;

Calderone & Fonzi, 2001; Sudbery et al., 2004; Chaffin, 2008).

The yeast-to-mycelium transition in C. albicans is linked

to its pathogenic nature. This is due in part to the fact that

newly formed filaments (germ tubes) are more adherent to

mammalian cells than are yeast cells (Odds, 1988; Cutler,

1991) and adherence certainly is a fundamental stage before

host tissue penetration. Genes that control hyphal morphol-

ogy are coregulated with genes that encode more standard

virulence factors such as proteases and adhesins (Pitarch

et al., 2006; Chaffin, 2008). Besides, hyphae are essential

elements for providing the structural integrity and multi-

layered architecture characteristic of mature/fully developed

biofilms, although yeast-only biofilms have also been de-

scribed (Baillie & Douglas, 1998, 1999). Candida albicans

strains with mutations in genes governing morphogenesis

and that are defective in filamentation also display defects in

their biofilm-forming abilities (Ramage et al., 2002; Garcıa-

Sanchez et al., 2004; Kelly et al., 2004; Krueger et al., 2004).

On the other hand, CSH contributes to the pathogenesis

of the opportunistic fungal pathogen C. albicans. Hydro-

phobic C. albicans cells are more adherent than hydrophilic

cells to a variety of host tissues, and the pattern of adherence

is more widespread (Hazen & Glee, 1995).

Finally, studies aimed at the identification of cell surface

components of C. albicans involved in the interaction of

fungal cells with host tissues have revealed the existence of a

large assortment of cell wall-bound carbohydrates such as

mannan, which has been shown to play an important role in

adhesion, host recognition and virulence (Calderone & Fonzi,

2001), and proteins displaying adhesin characteristics, such as

the glycosylphosphatidylinositol (GPI)-anchored species

(GPI-CWP) including the ALS gene family (Hoyer et al.,

2008), the CSA1, HYR1, HWP1 and EAP1 gene products

(Bailey et al., 1996; Staab et al., 1999; Lamarre et al., 2000;

Sundstrom et al., 2002; Li & Palecek, 2008), and a family of

surface-bound proteins containing an eight-cysteine domain

referred to as common in several fungal extracellular mem-

brane (CFEM), which may function as cell-surface receptors

or signal transducers, or as adhesion molecules in host–patho-

gen interactions (Kulkarni et al., 2003).

In a previous report (Perez et al., 2006), we described

several characteristics and functions of PGA10 (for pre-

dicted glycosylphosphatidylinositol-anchored), which is the

standard designation given by de Groot et al. (2003) to genes

coding for fungal glycosylphosphatidylinositol-anchored

proteins without a specific function. PGA10 gene (also

designated as RBT51; Weissman & Kornitzer, 2004) codes

for a putative member of the CFEM family, whose deletion

resulted in a cascade of pleiotropic effects, mostly affecting

cell surface-related properties (Perez et al., 2006). We also

examined the biofilm-forming ability, a feature that appears

to play a key role in virulence and pathogenesis in C. albicans

(Douglas, 2003; d’Enfert, 2006; Nett & Andes, 2006), of C.

albicans homozygous mutant strains harbouring single,

double and triple deletions for PGA10, as well as RBT5 and

CSA1 genes that also code for other CFEM proteins (Braun

et al., 2000; Lamarre et al., 2000), and found that these gene

products could be involved in the biogenesis and/or the

maintenance of biofilm structure and integrity in C. albicans

(Perez et al., 2006). In this paper, we have performed further

functional characterization of these mutant strains by ex-

amining CSH, relative gene expression profiling, cell surface

structure by atomic force microscopy (AFM), additional

structural features of biofilms and virulence in animal

models. The results reported in this work support the

contention for a role of the different proteins belonging to

the CFEM family present in C. albicans in the interaction of

fungal cells with the external environment (including bio-

film biogenesis), although they do not appear to be directly

involved in virulence. Consequently, further work is neces-

sary to fully elucidate all possible aspects of the biological

and functional role (for instance, to determine whether

these genes may represent potential biological targets for

new anti-Candida therapies) of this intriguing family of

proteins in C. albicans.

Materials and methods

Strains and growth conditions

The C. albicans strains used are listed in Table 1. Cells were

routinely grown in YPD [2% glucose, 1% yeast extract, 2%

Bacto peptone (Difco)] or YNB (0.67% yeast nitrogen base

without amino acids, 2% glucose) media at 28 1C with

shaking (100 r.p.m.). Media were supplemented with uri-

dine (25mg mL�1) when required.

Germ tube formation in C. albicans was induced using the

starvation method (Casanova et al., 1989). The formation of

biofilms by the different C. albicans strains was assessed using

the procedure described elsewhere (Ramage et al., 2001).

Briefly, cells were grown overnight in an orbital shaker in

YPD medium, harvested and washed in sterile 10 mM phos-

phate-buffered saline (PBS), pH 7.4. Cells were suspended in

Roswell Park Memorial Institute (RPMI)-1640 medium sup-

plemented with L-glutamine and buffered with 4-2-hydro-

xyethyl-1-piperazineethanesulfonic acid (Sigma Chemical Co.,

St. Louis, MO) to a final concentration of 1� 106 cells mL�1.

Biofilms were formed by pipetting appropriate volumes of the

standardized cell suspensions into wells of commercially

FEMS Yeast Res 11 (2011) 273–284c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

274 A. Perez et al.

available presterilized, polystyrene, flat-bottomed, 96-well

microtitre plates or cell tissue flasks (Nalge Nunc Interna-

tional, Denmark) and incubated at 37 1C.

Detection of PGA10 promoter activity by flowcytometry of green fluorescent protein (GFP)reporter expression

Plasmid construction was based on the procedure reported by

Barelle et al. (2004). The PGA10 promoter region was ampli-

fied by PCR using the primers 50-GCGCCTCGAGTGTTGAA

GAGCAGGCTATGG-30 and 50-GCGCAAGCTTGCGGATT G

ACTTGAAGAAAATC-30 (XhoI and HindIII sites underlined)

and cloned between the XhoI and the HindIII sites of pGFP.

The plasmid was linearized by digestion with BglII and used to

transform C. albicans, generating strain GAP3 (see Table 1).

The relative GFP fluorescence of GAP3 cells was visua-

lized using confocal scanning laser microscopy (CSLM)

analysis with an LSM 510 META laser scanning microscope

(Zeiss, Germany) attached to an Axioplan II microscope

(Zeiss).

The relative gene expression in C. albicans cells was

determined by flow cytometry. Strain GAP3 was grown

overnight in YPD medium at 28 1C with shaking. Cells were

collected by centrifugation and grown under yeast- and

hyphae-inducing conditions until an OD600 nm of 0.2 was

reached. Aliquots of 0.5 mL from each culture were inocu-

lated in 20 mL of fresh YPD and RPMI media and incubated

at 28 and 37 1C, respectively. Samples (10mL) were taken at

different time intervals (0, 0.5, 1, 1.5 and 2 h) and examined

by optical microscopy to ensure that only yeast forms and

early hyphal filaments were present in each of the cultures.

Cells were collected by centrifugation, washed twice in PBS

and analysed in a Modular Flow Cytometer (MoFlo, Beck-

man Coulter).

Gene expression analysis by quantitative reversetranscriptase (RT)-PCR

PGA10 gene expression in both morphological forms was

assessed by quantitative RT-PCR and compared with the

expression of the 18S housekeeping gene.

For this purpose, total RNA from yeast and hyphal forms

was isolated from C. albicans with TRizol reagent (Invitro-

gen, Paisley, UK) following the manufacturer’s protocol,

after homogenizing the cells for three periods of time (30 s

each) using a mini-beadbeater that intensely agitates the

sealed microcentrifuge vial containing cells and 0.5 mL of

glass beads.

For cDNA synthesis in a first step, a known concentration

(from 200 ng to 2 mg) of total RNA was mixed with 1 mL of

random hexamer pd(N)6 (2 mM) and water up to 7 mL. The

solution was denatured at 65 1C for 10 min and allowed to

cool on ice for 5 min. Subsequently, 0.5 mL of dNTPs

(0.2mM), 2 mL of Moloney Murine Leukemia Virus

(MMLV) 5� reaction buffer and 0.5 mL of MMLV reverse

transcriptase (Invitrogen) were added to each of the tubes

(10mL final volume) and incubated at 37 1C for 1 h and

subsequently at 75 1C for 10 min. The cDNA was stored at

� 70 1C until required.

Table 1. Candida albicans strains used in this work

Strain Genotype Parental strain Reference

SC5314 Wild-type Gillum et al. (1984)

CAI4 ura3D<limm434/ura3D<limm434 SC5314 Fonzi & Irwin (1993)

CAI4-URA3 ura3D<limm434/ura3D<limm434, RP10<URA3 CAI4 This work�

CAN1 ura3D<limm434/ura3D<limm434, Pga10D<

hisG/pga10<hisG RP10<URA3

CA3 This work

BCa18-2 ura3D<limm434/ura3D<limm434, rbt5D<

hisG/rbt5D<hisG-URA3-hisG

CAI4 Braun et al. (2000)

BCa17-4 ura3D<limm434/ura3D<limm434, wap1D<

hisG/wap1D<hisG-URA3-hisG

CAI4 Braun et al. (2000)

KC100 ura3D<limm434/ura3D<limm434, rbt5D<hisG/rbt5D<

hisG pga10D<hisG/pga10D<hisG-URA3-hisG

CAI4 Weissman & Kornitzer (2004)

KCU1 ura3D<limm434/ura3D<limm434, rbt5D<

hisG/rbt5D<hisG pga10D<hisG/pga10D<hisG

RP10<URA3

This work

KC171 ura3D<limm434/ura3D<limm434, rbt5D<

hisG/rbt5D<hisG pga10D<hisG/pga10D<hisG

ccc2D<hisG/ccc2D<hisG

wap1D<hisG/wap1D<hisG

ade2D<CaCCC2

CAI4 Weissman & Kornitzer (2004)

GAP3 CAI4 CAI4 derivative, pPGA10-GFP This work

�The CAI4-URA3 strain was kindly provided by Dr Gwyneth Bertram, School of Medical Science, University of Aberdeen.


275Candida albicans mutants for CFEM domain-containing proteins

For each qPCR reaction, 0.25mL of cDNA (1500 ng mL�1)

was mixed with 12.5mL SYBRs Green (Invitrogen), 0.5mL

Rox reference dye (Invitrogen), 0.5 mL each pair of primers

generating unique cDNA amplifications for PGA10 (50-CCA

CTACCTCCGACACCACT-30 and 50-TTCTGCTGCGGAG

GACTT-30) and 18s (50-GATTAGATACCCTGGTAG-30 and

50-ATTGTAGCACGTGTGTAG-30), 1.5mL of DNA Taq poly-

merase and distilled water to a final volume of 25mL.

All samples were prepared and processed in triplicate and

the mean Ct value was calculated. The cDNA levels during

the linear phase of amplification were normalized against

the 18S ribosomal housekeeping gene and used to calculate

the relative levels of expression using the 2�DDCt method.

All quantitative RT-PCR reactions were performed using

the DNA Engine OpticonTM 2qPCR machine (MJ Research,

Waltham, MA), and all optical reading data and Ct values

were stored and analysed using Opticon Monitor version

2.01 (MJ Research).

Evaluation of relative CSH by flow cytometry

Relative yeast CSH was determined following a method

developed by Colling et al. (2005), which is a variant of the

latex-polystyrene microsphere assay described by Hazen

& Hazen (1987) and Lopez-Ribot et al. (1991). Briefly, yeast

cells were suspended in PBS and diluted to an OD600 nm of

1.0� 0.1. Polystyrene beads (0.834mm, average diameter),

deep blue-dyed, were purchased from Sigma Chemical Co. A

beads suspension was provided in 1 mL of suspension (10%

solids). For bead adherence tests, a microsphere stock suspen-

sion was vigorously mixed and 10mL of this suspension was

added to a 1 : 100 dilution of a yeast cells suspension in PBS.

Beads and yeasts were allowed to interact at room temperature

for 30 min on a rotator at 14 cycles min�1. Subsequently, each

sample was vortexed vigorously and analysed by flow cytome-

try in a Beckman Coulter MoFlo cytometer.

AFM

For AFM examination, cells were grown overnight at 30 1C in

YPD medium, collected by centrifugation (1–5 mL of cell

culture), washed once in PBS and resuspended in about

100mL of distilled water. A drop of the resulting cell suspen-

sion was placed on a coverslip and allowed to dry completely.

Coverslips were placed in a Nanoscope IV/Dimension 3100

SPM from Veeco Metrology (Santa Barbara, CA), and the

experiments were conducted in the tapping mode for the

imagery, using a Phosphorous-doped Si cantilever (RTESP

Model, Veeco Metrology). The parameters for measurement

were as follows: Spring constant, �80 N m�1; resonance

frequency, �260 kHz; scan speed, 0.5 Hz; and scan range

varying from 500 nm to 10mm. Images were processed using

the NANOSCOPE IV version 5.30 software.

Determination of biofilm structure by CSLM

Biofilms formed by different mutant strains studied in this

work were further characterized by CSLM.

Cells were grown under biofilm-forming conditions as

described by Ramage et al. (2001) on ThermanoxTM coverslips

placed in sterile 12-well cell culture plates. After incubation for

48 h, coverslips were transferred to new cell culture plates,

gently washed with PBS and incubated in the dark for 30 min

in 1 mL of PBS containing FUN-1 (10mM) alone or a mixture

of FUN-1 (10mM) and Concanavalin A (ConA)-Alexa Fluor

488 (5mM) (Molecular Probes, Eugene, OR). FUN-1 is a

cytoplasmic fluorescent probe to assess cell viability, whereas

the ConA-Alexa Fluor 488 Conjugate selectively binds to

mannose and glucose residues present in polysaccharides,

which are major constituents of the cell wall and the extra-

cellular matrix (EM) of biofilms in C. albicans.

The CSLM analysis was performed using an LSM 510

META laser scanning microscope (Zeiss) attached to an

Axioplan II microscope (Zeiss). Biofilms were observed

using � 40 and � 100 oil immersion objectives. The excita-

tion wavelengths were 488 nm (Argon laser) and 543 nm

(He-Ne laser), and the emission wavelengths were 505 and

560 nm for Alexa Fluor 488 and FUN-1, respectively. To

determine biofilm structure, a series of horizontal (x–y axes)

optical sections were taken throughout the depth of the

biofilm (z axis). Three-dimensional representations showing

the relative fluorescence for each fluorophore in biofilms

formed by the different C. albicans strains were made using

the built-in software provided with the equipment.

Virulence assays

Cultures of the different strains were obtained by incubation

at 28 1C for 14–16 h in YPD medium. The average OD600 nm

determined using a spectrophotometer was found to be in

the range of 1.1–1.3 for all the cultures after the incubation

period (according to the OD600 nm values measured, all the

cultures were close to the late exponential growth phase),

which indicated a similar growth rate for all the different

strains tested. Cells were subsequently harvested by centri-

fugation, washed three times in a sterile pyrogen-free saline

solution and counted using a haemocytometer chamber.

Appropriate suspensions from the cultures to reach a final

concentration of 5.0� 105 cells mL�1 were prepared and

200 mL aliquots from these suspensions containing a total

infecting dosage of 105 cells were immediately injected into

the lateral tail veins of 6–8-week-old female BALB/c mice

(obtained from the National Cancer Institute).

Groups of six mice were used for each of the strains

tested, and were observed during a total of 28 days post-

infection. For statistical analysis, survival data and differ-

ences between groups were analysed using the Kaplan–Meier

and log-rank tests. All experiments were performed in


276 A. Perez et al.

accordance with Institutional regulations in an Association

for Assessment and Accreditation of Laboratory Animal

Care (AAALAC)-certified facility at the University of Texas

Health Sciences Center at San Antonio (UTHSCSA). Mice

were allowed a 1-week acclimatization period before the

experiments were started.

Results

Relative gene expression analysis using areporter strain

The relative gene expression within individual C. albicans

cells was assessed as described in Materials and methods.

The PGA10 promoter was inserted into the plasmid pGFP,

which contains the codon-optimized yeast enhanced green

fluorescent protein (yEGFP; Cormack et al., 1997), generat-

ing a promoter–GFP fusion (Fig. 1). Strain CAI4 of C.

albicans was transformed with plasmid pPGA10-yEGFP and

a new reporter strain, GAP3, was generated.

The activity of this GFP fusion was measured in indivi-

dual cells using fluorescence microscopy (Fig. 2a). GFP

fluorescence must increase relatively quickly following gene

activation, and decline relatively quickly once the gene is

repressed. Therefore, these reporter cells can also be used to

determine the relative gene expression by flow cytometry.

Cells corresponding to strains CAI4 and GAP3 were grown

under yeast- and hyphae-inducing conditions as described

in Materials and methods and analysed by flow cytometry.

As shown in Fig. 2b, there was an increase in fluorescence

(fluorescence channel 1) as the cells began to form hyphae,

which means that PGA10 is a gene whose expression is

induced under mycelium-forming conditions. These results

were confirmed by qRT-PCR analysis, which revealed that

the expression of the PGA10 gene increased up to 42 times in

mycelia cells compared with yeasts (Fig. 3). A similar over-

expression pattern under hyphae-inducing conditions was

reported earlier for some genes of the CFEM family (RBT5

and CSA1/WAP1) by Braun et al. (2000) and Lamarre et al.

(2000) and more recently by Sosinska et al. (2011), which

demonstrated that the protein level of Rbt5 was 10-fold

higher in the cell walls of filamentous cultures growing at

pH 7.0 compared with yeast cultures growing at pH 4.0.

Determination of CSH

CSH was determined as described above. Before quantifying

CSH in cells, the optimal working conditions for the assay

were established. Fig. 4a shows that fluorescence channel 4

(FL4) detected the blue-dyed microspheres alone, whereas

the control yeast cells did not show any fluorescence (Fig.

4b). After incubating the cells with the microspheres, the

fluorescence level that had a direct correlation with the

binding of the microspheres to the cells and therefore with

the CSH was measured using flow cytometry. Cells of all

mutant strains manifested similar CSH levels that were, in

all cases, higher than the CSH exhibited by the control

CAI4-URA3 parental strain (Fig. 4c–h). These higher CSH

values were in correlation with an increased ability of

adhesion to different serum and animal tissues proteins

(fibrinogen, laminin, fibronectin and EM) displayed by the

mutant strain (results not shown).

Cell surface examination by AFM

The phenotype observed, i.e. higher CSH values and an

increased ability of adhesion to different serum and animal

tissue proteins, were indicative of a defective cell wall

structure and/or composition in the mutant strains. In

addition, we have also reported that the null pga10D mutant

displayed an increased sensitivity to cell wall-perturbing

agents such as calcofluor white, Congo red and sodium

dodecyl sulphate (Perez et al., 2006). Consequently, we

considered it of interest to perform a visual examination of

the cell surface of C. albicans strains bearing single, double

and triple deletions for PGA10, RBT5 and CSA1 genes using

AFM, a novel and powerful tool that provides a topographi-

cal image of cell surface at a high magnification and allows

the determination of cell wall nanomechanical and func-

tional (including CSH) properties of yeasts (Dague et al.,

2010; Dufrene, 2010).

Images of the cells were taken in order to obtain informa-

tion regarding the morphological characteristics of the cell

surface for each strain examined. As can be observed in Fig.

5a–d, the parental CAI4-URA3 strain showed a smooth and

homogeneous cell surface, whereas the single mutants such

as pga10D, rbt5D and csa1D showed a highly heterogeneous

and rougher cell surface that was clearly noticeable in the

case of cells from strain csa1D. Alterations of the cell surface

were dramatically apparent in the case of the strains bearing

Fig. 1. Structure of the pPGA10-yEGFP reporter plasmid. The vector

was linearized with BglII before the integration into the Candida albicans

CAI4 strain.



double and triple gene knockouts such as pga10D/rbt5D and

pga10D/rbt5D/csa1D (Fig. 5e and f).

Biofilm structure analysis by CSLM

The cell surface plays a key role in the initial interaction of C.

albicans cells with an inert or a biological substrate in the

process of biofilm formation. We have reported previously

that C. albicans mutants with single, double and triple

deletions for genes encoding proteins species bearing the

CFEM domain (Pga10p, Rbt5p and Csa1p) exhibited an

abnormal ability to form biofilms (Perez et al., 2006), and in

this communication, we reported that the surface of mutant

strains displayed severe morphological alterations. Conse-

quently, we considered it of interest to further inspect the

biofilm three-dimensional structure and development by

means of CSLM. This technique was preferred to scanning

electron microscopy because it is a nondestructive technique

that maintains the biofilm under native conditions with

minimal structural alterations, allowing for the in situ

visualization of the mature biofilms.

For this purpose, a combination of FUN-1, a fluorescent

dye taken up by fungal cells that, in the presence of

metabolic viability, is converted from a diffuse yellow

cytoplasmic stain to red, rod-like collections, and ConA

conjugated with the dye Alexa Fluor 488, which binds

specifically to polysaccharides including a-mannopyranosyl

and a-glucopyranosyl residues and yields green fluorescence

(Kuhn et al., 2002), was used.

Examination by CSLM of mature (24 h) biofilms stained

with FUN-1 revealed that biofilms formed by mutant strains

had only a 20–30% depth compared with the biofilm

belonging to the control strain CAI4-URA3. Thus, the depth

of biofilms formed by the parental strain was 200� 20 mm,

Fig. 2. PGA10 expression analysis using a reporter strain. CSLM analysis revealed higher fluorescence levels in biofilms formed by GAP3 strain (see Table

1) (mostly hyphal filaments) compared with planktonic (free-floating forms) yeast cells (a). Studies of PGA10 expression by flow cytometry (b) revealed

that fluorescence corresponding to the GAP3 strain is increased under hyphae-inducing conditions (green line), whereas it remains stable under yeast-

forming conditions (pink line). Strain CAI4 was used as a negative fluorescence control.

Yeast Mycelia0.000

0.005

0.010

0.015

0.020 0.0187

0.00044Exp

ress

ion

rel

ativ

e to

HK

gen

e

Fig. 3. Evaluation of PGA10 gene expression by qRT-PCR. The relative

expression of PGA10 was analysed in yeast and hyphae using the

endogenous 18S ribosomal gene transcript as a housekeeping (HK)

reference. Based on the levels of relative expression, yeast cells expressed

42.52 times less PGA10 (0.00044) than hyphae (0.0187) under the

conditions tested. The amplification efficiencies of PGA10 were shown

to be similar under both conditions.


278 A. Perez et al.

whereas in the case of the different mutants examined,

biofilm depths ranged from 40� 11 to 60� 17mm (figures

were the mean values of five independent measures of

biofilm depth in three different areas for each of the strains

examined). These results indicated that biofilms produced

by the mutants contained a lower proportion of metaboli-

cally active cells, which in turn may account for a lower

thickness of mature biofilms, a contention that was addi-

tionally supported by subsequent CSLM observations of

biofilms double stained with FUN-1 (red fluorescence) and

ConA Alexa Fluor 488 (green fluorescence). Thus, a com-

parison of the relative fluorescence levels due to both

fluorophores revealed that biofilms formed by mutant

strains contained not only a lower proportion of metaboli-

cally active cells but also a lower quantity of exopolymeric

material, compared with the parental CAI4-URA3 strain

(Fig. 6). In any case, the mutant strains retained the ability

to form hyphae in the biofilms, because similar relative

proportions of mycelial forms were consistently observed

(when compared with the parental CAI4-URA3 strain).

Virulence assessment

The abilities to grow as mycelia filaments and to form

biofilms are believed to be important virulence traits in C.

albicans. Besides, hyphae are the most abundant cellular

elements present in candidal biofilms. We have found that

proteins belonging to the CFEM family are overexpressed

under hyphae-inducing conditions and mutants for genes

coding for such proteins are defective in biofilm formation.

Consequently, a possible role for CFEM proteins in C.

albicans virulence could be expected. In order to assess such

a contention, virulence studies were performed in animal

models. Female BALB/c mice were infected with 200 mL of

cell suspensions containing 5.0� 105 cells mL�1 (the total

infecting dosage administered was 105 cells). The number

Fig. 4. Measure of relative CSH. The Candida

albicans parental CAI4-URA3 strain did not show

fluorescence on the channel FL4 (a), whereas the

blue-dyed polystyrene beads showed a very

strong fluorescence (b). After incubating the cells

with the microspheres, the fluorescence level

was measured using a flow cytometer. The con-

trol strain cells CAI4-URA3 (c) presented a lower

binding degree of microspheres to the cell sur-

face compared with the mutant strains csa1D (d),

pga10D (e), pga10D/rbt5D (f), rbt5D (g), y

pga10D/rbt5D/csa1D (h).



and viability of cells present in the infecting inocula

were further assessed by a plate count in YPD agar. In all

cases, the plate count yielded lower values [CAI4-URA3

(3.8� 105 cells mL�1); pga10D (3.3� 105 cells mL�1);

pga10D/rbt5D (4.1� 105 cells mL�1); and pga10D/rbt5D/

csa1D (2.4� 105 cells mL�1)] when compared with the re-

sults obtained by direct counting with the haemocytometer

chamber. This lack of correlation seemed to be most likely

due to the clumping tendency of C. albicans cells (clumps

are extremely difficult to disintegrate when cell suspensions

are mixed with the melted YPD agar before pouring into the

Petri dishes), which was found to be particularly high in the

case of the triple homozygous mutant (pga10D/rbt5D/

csa1D) (Perez et al., 2006), rather than to a reduction in the

number of viable cells since cultures were harvested before

they reach the late exponential growth phase (see Materials

and methods), a phase in which most, if not all, cells are

viable and metabolically active.

Figure 7 shows that the virulence of pga10D and pga10D/

rbt5D mutants was similar to that displayed by the control

CAI4-URA3 strain. In the case of the triple mutant pga10D/

rbt5D/csa1D strain, the lack of virulence could be a con-

sequence of the wrong localization of the URA3 gene. The

right orientation and location of the gene URA3 has been

shown to affect C. albicans virulence (Cheng et al., 2003;

Brand et al., 2004). Under our experimental conditions, we

Fig. 5. AFM observations at two scan ranges of

the surface of Candida albicans yeast cells from

CAI4-URA3, pga10D, rbt5D, csa1D, pga10D/

rbt5D and pga10D/rbt5D/csa1D strains. All single

mutant strains presented a highly heterogeneous

and rougher cell surface when compared with

the control CAI4-URA3 strain. These alterations

of the cell surface were dramatically apparent in

the case of the double and triple mutant strains.

Fig. 6. Relative fluorescence associated with

biofilms formed by strains CAI4-URA3 (a),

pga10D (b), rbt5D (c), csa1D (d), pga10D/rbt5D(e) y pga10D/rbt5D/csa1D (f) on ThermanoxTM

coverslips, double stained with FUN-1s

(red fluorescence) and Con-A Alexa fluor 488

(green fluorescence). Results from CSLM

visualization are shown as a three-dimensional

reconstruction plot representing the intensity of

fluorescence for each fluorophore. Colour

images were produced by pasting each of the

greyscale images (the scale values for apparent

brightness in x, y and z axes range from 0 to

250) from the confocal microscope using the

built-in software of the equipment.


280 A. Perez et al.

have found that the effect of URA3 seems to be variable.

Thus, in the case of the pga10D mutant, the lack of URA3

resulted in loss of virulence (CAI4-URA3 vs. pga10D-

URA3�; results not shown), whereas reintegration of URA3

restores its virulent nature (Fig. 7). However, we found that

a csa1D null mutant strain in which the URA3 was not

reintegrated exhibited a virulence very similar to the par-

ental CAI4-URA3 strain (P = not significant for CAI4-URA3

vs. csa1D-URA3�; results not shown). In any case, we

replaced the URA3 gene in the RPS10 locus using the

integration vector CIp10 (Murad et al., 2000) in both the

single (pga10D) and the double (pga10D/rbt5D) homozy-

gous mutant strains before testing virulence. However,

several attempts to achieve the reintegration of the URA3

gene in its right locus in the triple mutant were unsuccessful

and, therefore, virulence assays ought to be carried out with

the original strain (Weissman & Kornitzer, 2004). Conse-

quently, it seems that further investigation is required to

elucidate not only the molecular basis of the avirulent

nature displayed by the triple mutant but also the actual

role of URA3 in C. albicans virulence.

Discussion

Most of pathogenic fungal species known carry a large

number of species belonging to the family of the CFEM

domain (Kulkarni et al., 2003), which suggests a potential

role of these proteins in pathogenesis. Six CFEM-containing

proteins that are the products of RBT5, CSA1 (also desig-

nated as WAP1), CSA2, PGA7, SSR1 and PGA10 genes have

been currently identified in C. albicans (Braun et al., 2000;

Lamarre et al., 2000; de Groot et al., 2003; Weissman &

Kornitzer, 2004; Garcera et al., 2005; Castillo et al., 2008;

Weissman et al., 2008).

We have previously examined some biological features of

strains carrying single, double and triple deletions for

PGA10, RBT5 and CSA1. The single null mutant for the

PGA10 gene was generated in our laboratory (Perez et al.,

2006). The rest of the mutants examined were kindly

provided by other groups (Braun et al., 2000; Weissman &

Kornitzer, 2004). All these mutants formed fragile biofilms

in vitro, with a low adherence to the substrate (Perez et al.,

2006). This suggests that CFEM-containing proteins are

involved in the biogenesis of biofilms in C. albicans. These

results are in agreement with reports from other laboratories

indicating that several GPI–CWP species including Als1p,

Als3p, Hwp1p and Eap1p appear to be required for biofilm

formation in C. albicans (Firon et al., 2007; Hiller et al.,

2007; Li et al., 2007; Nobile et al., 2008). To some extent,

biofilms formed by strains bearing deletions in genes coding

for members of the CFEM family phenocopy those formed

by als1D/als3D and hwp1D mutants (Nobile et al., 2008).

In this work, we have further examined biofilms formed by

mutant strains for the CFEM domain by means of CSLM

using a combination of the fluorescent dyes FUN-1 and

ConA-Alexa Fluor 488 Conjugate (see Materials and meth-

ods), and found that such biofilms displayed a thin three-

dimensional structure with 40–60mm depth, which repre-

sented only 20–30% thickness of the biofilm formed by the

control strain CAI4-URA3, with lower quantities of the EM

component and also a decrease in the quantity of metaboli-

cally active cells. These observations strongly suggest that the

deletion of genes coding proteins belonging to the CFEM

family is associated with a defect in the general structure of

biofilms. The biofilm-deficient phenotype could be a non-

specific effect of the absence of one or more CFEM proteins or,

alternatively, due to the fact that these species are putative cell

surface-bound components involved in the interaction of

fungal cells with the environment. In any case, the abnormal

biofilm-forming abilities observed do not appear to be related

to defects in filamentation because the mutant strains retained

unaltered their ability to form hyphae and similar relative

proportions of mycelial forms were consistently observed in all

biofilms examined in this work. In this context, it has been

clearly established that hyphae are essential elements for

providing the structural integrity and multilayered architec-

ture characteristic of mature/fully developed biofilms (Baillie

& Douglas, 1999), because C. albicans strains with mutations

in genes governing morphogenesis and that are defective in

filamentation also display defects in their biofilm-forming

abilities (Ramage et al., 2002; Garcıa-Sanchez et al., 2004; Kelly

et al., 2004; Krueger et al., 2004).

On the other hand, it has been suggested that CFEM

proteins may have a function as adhesion molecules in

host–pathogen interactions (Kulkarni et al., 2003).

Fig. 7. Survival/death graph of mice infected in vitro with the control

strain CAI4-URA3 and with the single (pga10D), double (pga10D/rbt5D)

and triple (pga10D/rbt5D/csa1D) null mutants. Statistically significant

differences (P-values) were found to be as follows: CAI4-URA3 vs.

pga10D, P = 0.0018; CAI4-URA3 vs. pga10D/rbt5D, P = 0.0070; CAI4-

URA3 vs. pga10D/rbt5D/csa1D, P = 0.0008.



Therefore, strains carrying deletions in any of the genes

coding for these proteins would be expected to display

altered cell surface properties (i.e. CSH and/or adhesiveness

to inert or biological surfaces and substrates) related to

interactions of fungal cells with the external environment.

Thus, mutant strains exhibited higher CSH levels and

increased binding ability to all different substrates tested.

Because hydrophobic C. albicans cells are more adherent

than their hydrophilic counterparts to a variety of proteins

from host tissues (Masuoka et al., 1999; de Repentigny et al.,

2000), the observations reported here suggest that the loss of

functionality of genes coding for CFEM domain-containing

proteins may result in a cascade of pleiotropic effects

including (1) overexpression of genes coding for other

proteins having adhesive properties or that confer CSH to

compensate the absence of CFEM species or (2) structural

cell surface alterations [CFEM mutants displayed higher

sensitivity to cell wall-disturbing agents, which suggests

possible alterations in the fungal cell wall structure and/or

function of these mutants (Perez et al., 2006)]. AFM

observations reported in this work strongly support this

latter contention because the cell wall surface of mutant

strains was found to display a very rough and disrupted

appearance when compared with the parental strain.

Although only yeast cells were examined by AFM, one can

speculate on the possibility that the phenotype observed

under AFM should be much more pronounced in hyphal

cells because, as above stated, some genes of the CFEM

family are overexpressed under hyphae-inducing conditions

(Braun et al., 2000; Lamarre et al., 2000; Sosinska et al.,

2011) and, consequently, knockout of such genes could be

associated with stronger cell surface alterations, taking into

account that the cell wall in hyphal filaments appears to be

thinner than that present in yeast cells (Rico et al., 1991).

Another interesting observation reported here is that the

PGA10 gene was found to be upregulated under hyphae-

inducing conditions, which has also been reported for other

genes (RBT5 and CSA1) of the CFEM family (Braun et al.,

2000; Lamarre et al., 2000). Because the ability to grow as

mycelia filaments is believed to be an important virulence

trait in C. albicans and hyphae are the most abundant

cellular elements present in candidal biofilms, proteins

belonging to the CFEM family may be expected to play an

important role in the virulence of this fungal species.

However, we found that strains bearing mutations in genes

coding for proteins belonging to the CFEM family were not

defective in animal models, thus suggesting that individu-

ally, neither of these genes were important to successfully

infect the host. Moreover, mutant strains exhibited a pattern

of susceptibility/resistance to antifungals similar to that

displayed by the parental strain (data not shown).

Other roles, for example haem–iron utilization and

haemin-binding capacity, have been suggested for PGA10

and RBT5 (Weissman & Kornitzer, 2004; Weissman et al.,

2008). Although none of the other members of the CFEM

family in C. albicans or in other fungal species have been

reported to share this function, the possibility that the

different phenotypes displayed by the CFEM mutant strains

examined in this and previous work from our group (Perez

et al., 2006) could be due to a defective iron uptake cannot

be completely dismissed. Hence, the question is whether

Pga10p and Rbt5p are the first proteins belonging to the

CFEM family to be assigned to a different specific function

or, in fact, they act as multifunctional proteins (Nombela

et al., 2006) that can display several functions depending on

their localization in the cell. Although CFEM-containing

proteins are believed to play a role in pathogenesis, acting as

cell-surface receptors or signal transducers, as adhesion

molecules in host–pathogen interactions in C. albicans or

in biofilm formation, further work is necessary to fully

elucidate all possible aspects of the biological and functional

role (including their potential as targets for new therapeutic

approaches for Candida infections) of this intriguing family

of proteins in C. albicans.

Acknowledgements

This work was supported by grants BFU2005-02572, Minis-

terio de Educacion y Ciencia, Spain, and ACOMP06/103,

Generalitat Valenciana, Valencia, Spain (to J.P.M.). A.P. was

the recipient of a predoctoral grant from Ministerio de

Educacion y Ciencia, Spain. We acknowledge D. Kornitzer

(Haifa, Israel) and A.D. Johnson (San Francisco, CA) for the

kind gift of the single, double and triple C. albicans mutant

strains for the RBT51, RBT5 and CSA1 genes. We thank John

Graham and Mahesh Uttamlal (Glasgow, UK) for their advice

with the AFM experiments, and Anna Lazzell (San Antonio,

TX) for her assistance with the virulence assays in mice.

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