Development of oxidative stress tolerance resulted in reduced ability to undergo morphologic transitions and decreased pathogenicity in a t-butylhydroperoxide-tolerant mutant of Candida

Developmentofoxidativestresstoleranceresulted in reducedabilitytoundergomorphologic transitionsanddecreasedpathogenicity ina t -butylhydroperoxide-tolerantmutantofCandidaalbicansAndrea Fekete1,2, Tamas Emri1, Agnes Gyetvai1, Zoltan Gazdag3, Miklos Pesti3, Zsuzsa Varga4,Jozsef Balla4, Csaba Cserhati5, Levente Emo+dy6, Lajos Gergely2 & Istvan Pocsi1

1Department of Microbial Biotechnology and Cell Biology, Faculty of Science, University of Debrecen, Debrecen, Hungary; 2Department of Medical

Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; 3Department of General and Environmental

Microbiology, Faculty of Science, University of Pecs, Pecs, Hungary; 41st Department of Medicine, Medical and Health Science Center, University of

Debrecen, Debrecen, Hungary; 5Department of Solid State Physics, Faculty of Science, University of Debrecen, Debrecen, Hungary; and 6Department of

Medical Microbiology and Immunology, Medical School, University of Pecs, Pecs, Hungary

Correspondence: Istvan Pocsi, Department

of Microbial Biotechnology and Cell Biology,

Faculty of Science, University of Debrecen, PO

Box 63, H-4010 Debrecen, Hungary. Tel.: 100

36 52 512900; ext. 62063; fax: 100 36 52

454400; e-mail: [email protected]

Received 24 August 2006; revised 15 February

2007; accepted 8 March 2007.

First published online 10 May 2007.

DOI:10.1111/j.1567-1364.2007.00244.x

Editor: David Goldfarb

Keywords

Candida albicans ; oxidative stress;

t -butylhydroperoxide; virulence attributes;

respiration; antimycotics.

Abstract

We tested the hypothesis that adaptation of Candida albicans to chronic oxidative

stress inhibits the formation of hyphae and reduces pathogenicity. Candida

albicans cells were exposed to increasing concentrations of t-butylhydroperoxide

(tBOOH), a lipid peroxidation-accelerating agent, and mutants with heritable

tBOOH tolerance were isolated. Hypha formation by the mutants was negligible

on Spider agar, indicating that the development of oxidative stress tolerance

prevented Candida cells from undergoing dimorphic switches. One of the

mutants, C. albicans AF06, was five times less pathogenic in mice than its parental

strain, due to its reduced germ tube-, pseudohypha- and hypha-forming capability,

and decreased phospholipase secretion. An increased oxidative stress tolerance

may therefore be disadvantageous when this pathogen leaves blood vessels and

invades deep organs. The AF06 mutant was characterized by high intracellular

concentrations of endogenous oxidants, reduced monounsaturated and polyunsa-

turated fatty acid contents, the continuous induction of the antioxidative defense

system, decreased cytochrome c-dependent respiration, and increased alternative

respiration. The mutation did not influence growth rate, cell size, cell surface,

cellular ultrastructures, including mitochondria, or recognition by human poly-

morphonuclear leukocytes. The selection of oxidative stress-tolerant respiratory

Candida mutants may also occur in vivo, when reduced respiration helps the

fungus to cope with antimycotic agents.

Introduction

Commensally growing Candida albicans (Mavor et al., 2005)

is more resistant to oxidative stress than the yeasts Sacchar-

omyces cerevisiae (Jamieson et al., 1996) and Schizosacchar-

omyces pombe (Smith et al., 2004), and is able to adapt to

oxidative stress caused by different oxidants in vitro (Jamie-

son et al., 1996). Candida albicans produces powerful

antioxidants to cope with reactive oxygen species (ROS)

(e.g. superoxide, peroxide, and hypochlorite) and reactive

nitrogen intermediates (RNIs) [e.g. nitric oxide (NO) and

peroxynitrite] produced by polymorphonuclear leukocytes

(PMNLs) and macrophages when fungal cells enter the

bloodstream and infect deep organs (Vazquez-Torres &

Balish, 1997; Mavor et al., 2005). The elements of antiox-

idative defense include small molecular mass metabolites

with high ROS quenching potential, e.g. D-erythroascorbic

acid (Huh et al., 2001) and 2,4-(hydroxy)phenyl-ethanol

(Cremer et al., 1999), as well as powerful enzymes neutraliz-

ing both ROS (e.g. catalase, glutathione peroxidases, super-

oxide dismutases, thioredoxin, thioredoxin reductase, and

methionine sulfoxide reductase) and RNIs (e.g. NO-respon-

sive flavohemoglobin) (Enjalbert et al., 2003; Ullmann et al.,

2004; Fradin et al., 2005; Hromatka et al., 2005). The

upregulation of this enzyme system has been demonstrated

in C. albicans cells exposed to whole blood and separated

PMNLs (Fradin et al., 2003, 2005) as well as to macrophages

(Lorenz et al., 2004).

FEMS Yeast Res 7 (2007) 834–847c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Although antioxidative enzymes of fungal pathogens are

regarded as ‘persistence factors’ (Mavor et al., 2005) pro-

moting the survival of these microorganisms during coloni-

zation and invasion rather than virulence attributes sensu

stricto (Hamilton & Holdom, 1999), the significance of their

activities and regulation cannot be underestimated, espe-

cially during penetration into deeper tissue and the blood-

stream, which is a rapidly changing and hostile environment

(Hube, 2004). Candida albicans does possess transcriptional

programs, including the fine-tuning of antioxidative defense

that enables this pathogen to resist the continuous attacks of

the host’s immune system (Lorenz & Fink, 2001; Fradin

et al., 2003, 2005; Lorenz et al., 2004; Smith et al., 2004;

Enjalbert et al., 2006). It is therefore understandable that

the disruption of genes coding for key enzymes in antiox-

idative defense may slow down growth [e.g. copper- and

zinc-containing superoxide dismutase (SOD1) (Hwang

et al., 2002), moderate virulence (e.g. SOD1) (Hwang et al.,

2002), catalase (Wysong et al., 1998), and YHB1 flavohemo-

globin (Hromatka et al., 2005)] and may reduce viability in

the presence of whole blood and PMNLs [e.g. SOD5

glycosylphosphatidylinositol-anchored SOD (Fradin et al.,

2005)].

Previously, it has been reported that the induction of

oxidative stress responses by exposure of C. albicans to

immune system cells inhibits the development of hyphae

(Fradin et al., 2003, 2005), an important virulence attribute

that facilitates escape of C. albicans from the bloodstream

and subsequent invasion of tissues (Calderone & Fonzi,

2001; Mavor et al., 2005). To test the hypothesis that

adaptation to chronic oxidative stress would inhibit forma-

tion of hyphae and reduce pathogenicity, C. albicans was

exposed to increasing concentrations of t-butylhydroperox-

ide (tBOOH), an oxidative stress-generating agent that has

long-lasting physiologic effects (Emri et al., 1999) and

accelerates lipid peroxidation chain reactions in biological

membranes (Greenley & Davies, 1992). This hydroperoxide

was selected because oxidative injuries of biological mem-

branes caused by tBOOH and the phagocytes’ NADPH

oxidase–myeloperoxidase (MPO) system may be quite simi-

lar. The NADPH oxidase–MPO system generates versatile

ROS, OCl�, tyrosyl radical and nitrating intermediates

(Brennan et al., 2001), which effectively modify and oxidize

lipids through lipid peroxidation pathways similar to

tBOOH (Savenkova et al., 1994; Byun et al., 1999). Impor-

tantly, the physiologic and transcriptional effects of tBOOH

and H2O2, a toxic decomposition product of superoxide

produced by phagocyte NADPH oxidases, seemed to be

equivalent in C. albicans in previous oxidative stress re-

sponse and sensitivity studies (Singh et al., 2004; Smith

et al., 2004; Enjalbert et al., 2006).

A series of C. albicans strains with heritable tolerance to

tBOOH were isolated and characterized in terms of both

morphology and physiology. Consistent with our hypoth-

esis, oxidative stress responses were continuously upregu-

lated in the mutant strains with reduced ability to form

hyphae and, concomitantly, with a significant reduction in

pathogenicity. A further hypothesis concerning the possible

in vivo development of C. albicans cells simultaneously

tolerant to chronic oxidative stress and antimycotics is also

presented and discussed here.

Materials and methods

Organisms, culture conditions, development oftBOOH-tolerant mutants, inheritability oftBOOH tolerance, colony size and hypha-formingcapability

tBOOH-tolerant C. albicans strains were developed from

C. albicans ATCC 14053, which was used as the control

strain in physiologic experiments and virulence studies.

Mutants and the control strain were maintained on Sabour-

aud dextrose agar (SDA) slopes [2% (w/v) glucose, 1% (w/v)

peptone, 2% (w/v) agar; pH 5.6], and slope cultures not

older than 1 week were used in further experiments.

Under standard yeast propagation conditions, C. albicans

was grown in 5-mL aliquots of Sabouraud dextrose broth

(SDB) [2% (w/v) glucose, 1% (w/v) mycological peptone;

pH 5.6] for 17 h in a rotary incubator at 28 1C. Unless

otherwise indicated, shake flasks (volume = 100 mL)

containing SDB (20 mL) were inoculated from over-

night standard cultures and incubated in an orbital shaker

at 28 1C and 140 r.p.m. (Gyetvai et al., 2006). The starting

OD was always set to 0.1 (l= 640 nm), and the cultures

were supplemented with 2–16 mmol L�1 tBOOH as

required.

tBOOH-tolerant mutants were developed by continuous

cultivation of C. albicans ATCC 14053 in the presence of

stepwise increasing concentrations of tBOOH under condi-

tions similar to those used by Fekete-Forgacs et al. (2000) for

the development of fluconazole-tolerant mutants. Briefly,

a 20-mL C. albicans ATCC 14053 shake flask culture was

grown for 10 h and exposed to 2 mmol L�1 tBOOH for 14 h.

Following this, tBOOH-treated C. albicans cells were sub-

cultured three times for 24 h in freshly prepared SDB

medium supplemented with 2 mmol L�1 tBOOH. In the

next set of experiments, yeast cells from the third subculture

were incubated for 10 h in the presence of 2 mmol L�1

tBOOH, and the concentration of tBOOH was then in-

creased to 4 mmol L�1 for another 14 h of incubation. After

three subculturing steps, the same procedure was repeated at

8 and 16 mmol L�1 tBOOH, always using the third subcul-

tures to inoculate the next sets of tBOOH exposure experi-

ments. Candida albicans cells from the third subcultures

were also plated onto SDA, and single colonies were isolated

FEMS Yeast Res 7 (2007) 834–847 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

835Oxidative stress tolerance and morphology in C. albicans

and cultured in tBOOH-free SDB. All mutants were identi-

fied as C. albicans using both the API ID32C kit (bioMer-

ieux) and CHROMagar Candida isolation and identification

medium (Becton Dickinson).

To check the reproducibility of the development of

tBOOH-tolerant mutants, the mutant generation and selec-

tion procedure was repeated 10 times (experiments 1–10),

starting from C. albicans ATCC 14053. The development

and inheritability of tBOOH tolerance were monitored by

measuring minimal inhibitory concentration (MICtBOOH)

values and specific glutathione reductase (GR) activities.

The inheritability of tBOOH tolerance in C. albicans

mutants isolated after exposure to 8 mmol L�1 tBOOH

(C. albicans AF01–10 strains, from experiments 1–10, re-

spectively) was demonstrated by comparing MICtBOOH and

specific GR activity values determined before and after 10

subsequent passages on SDA plates.

To screen for the formation of petite mutations under

chronic oxidative stress, aliquots of cell suspensions (50mL

each) containing 102 cells (C. albicans AF01–10 or ATCC

14053) were spread on SDA plates in 10 replicates, and the

colony sizes were compared in 2-, 3- and 5-day cultures.

The hypha-forming capability of the C. albicans AF01–10

and ATCC 14053 strains was compared by spotting 5� 104

yeast cells in 5-mL aliquots onto Spider agar medium

[1% (w/v) nutrient broth, 1% (w/v) glucose, 0.2% (w/v)

K2HPO4, and 1.35% (w/v) agar], incubating the cultures at

28 1C for 10 days, and observing hypha formation micro-

scopically.

For detailed physiologic, morphologic and virulence

studies, the C. albicans AF06 tBOOH-tolerant mutant

(Strain Collection of the Department of Microbiology and

Biotechnology, University of Debrecen) was selected after

8 mmol L�1 tBOOH treatments in the sixth series of mutant

generation experiments. Candida albicans AF06 showed an

average tBOOH tolerance among the C. albicans mutants

isolated after 8 mmol L�1 tBOOH treatments.

tBOOH tolerance, antigenicity, andpathogenicity

The continuous induction of the antioxidative defense

system in C. albicans and the concomitant reduction in the

hypha-forming capability of the fungus may adversely affect

its virulence. Therefore, the virulence attributes, antigenicity

and pathogenicity of the C. albicans AF06 mutant and its

parental strain C. albicans ATCC 14053 were tested and

compared.

Virulence attributes

Germination capability, secretion of aspartic protease and

phospholipase acivities were measured as described else-

where (Gyetvai et al., 2007). The hypha-forming capabilities

of the strains were estimated as described above. The

formation of pseudohyphae and chlamydospores by

C. albicans parental and mutant strains was observed

microscopically. Yeast cells collected from standard 17-h

cultures of C. albicans AF01–10 and ATCC 14053 by

centrifugation (1800 g, 10 min, 4 1C) were washed three

times with phosphate-buffered saline (PBS) and resus-

pended in PBS (107 cells mL�1). Five-microliter aliquots of

this suspension were spotted onto corn-meal agar [0.19%

(w/v) corn-meal agar, 1% (w/v) Tween-80], the spots were

covered with a sterile microscopic coverslide, and the

cultures were incubated at 28 1C for 62 h.

Antigenicity

The antigenicity of C. albicans AF06 and ATCC 14053 cells

was characterized by the amount of superoxide produced by

PMNLs in the presence of opsonized C. albicans cells

(Gyetvai et al., 2007). Opsonized yeast cells were combined

with PMNL suspension at a ratio of 1 : 100–1 : 25, and

superoxide production after 60 min of incubation at 37 1C

was assessed spectrophotometrically by measuring the re-

duction of cytochrome c (Babior et al., 1973) in a microassay

(Varga et al., 2001). Experiments were performed using

PMNL preparations from three healthy individuals, and

were performed in triplicate.

Pathogenicity

Female NMRI mice of SPF hygienic category (Charles River

Ltd, Budapest, Hungary), weighing 13–17 g, were injected

with 0.5-mL aliquots of C. albicans cell suspensions contain-

ing 1� 107, 2� 106, 4� 105, 8� 104, 1.6� 104 and 3.2� 103

cells parenterally into the tail vein. The survival of the mice

was monitored daily for 2 weeks, and the LD50 values for the

mutant and control strains were calculated using the Spear-

man–Karber method (Karber, 1931). In intraperitoneal

mouse assays, mice were injected with 106 C. albicans cells.

Groups of four mice each were sacrificed at 4 and 24 h after

infection, and the peritoneal cavities were rinsed with 1 mL

of PBS. Colony-forming C. albicans cells were counted in

rinsing fluids by plating series of dilutions onto SDA. At the

same time, the kidneys were removed, fixed in formalin, and

embedded in paraffin. Histologic sections were prepared

and stained by the periodic acid Schiff technique (Frankel

et al., 1970) to visualize C. albicans cells. The sections were

evaluated for the presence and morphology of fungal

structures.

t BOOH tolerance and cell morphology andphysiology

In order to obtain a deeper insight in the development of

tBOOH tolerance in C. albicans, the morphologic and


836 A. Fekete et al.

physiologic characteristics of the AF06 mutant and its

parental strain were compared.

Cell size, cellular ultrastructures andmitochondrial function

A series of electron microscopic and light microscopic

studies was carried out to map morphologic and physiologic

changes in the AF06 mutant. Altered cell size, cell surface

and mitochondrial morphology and functioning are often

indicative of severe cell injuries in stress-exposed Candida

cells (Yang et al., 1998; Pas et al., 2004; Alviano et al., 2005).

For scanning electron microscopy studies, cells from 17-h

standard cultures were suspended in 0.1 mol L�1 potassium

phosphate buffer (pH 7.0) at a cell density of 106 cell mL�1.

Cells were fixed with 2.5% glutaraldehyde solution and 1%

osmium tetroxide, and were drained with ethanol/water

using increasing ethanol concentrations according to Ara-

ncia et al. (1995). The cell preparations were dried in a

desiccator and coated with gold, and photomicrographs

were taken with an AMRAY 1830-I scanning electron

microscope (Amray Inc. Bedford, MA). Automatic particle

size analysis was implemented in the LABVIEW IMAQ VISION

software system (National Instruments, Austin, TX).

For transmission electron microscopy studies, C. albicans

cells from standard cultures were transferred into ice-cold

PBS, and the cell density was adjusted to 106 cell mL�1.

Yeast cells were stained with freshly prepared 2% KMnO4

solution for 1 h at room temperature, and washed

with distilled water according to Johnson et al. (1973).

Photomicrographs were taken with a JEOL Jam-1010

transmission electron microscope (JEOL, Tokyo, Japan),

and cell wall structures, nuclei and mitochondria of

C. albicans AF06 and ATCC 14053 strains were compared

visually.

To test mitochondrial morphology and organelle func-

tioning, 10-mL aliquots of minimal medium[1% (w/v)

glucose, 0.5% (w/v) (NH4)2SO4, 0.25% (w/v) KH2PO4,

0.05% (w/v) MgSO4 � 7H2O, and 1% (v/v) Wickerham

solution] were inoculated with 17-h C. albicans cultures

(starting OD640 nm = 0.1) and incubated for 4 or 8 h at 28 1C.

Mitochondria were stained by incubating 1-mL aliquots of

these cultures with MitoTracker Red dye [0.1mmol L�1; an

indicator of the inner mitochondrial transmembrane poten-

tial (Macho et al., 1996); Molecular Probes, Eugene, Ore-

gon] for 30 min. MitoTracker Red-treated C. albicans cells

were separated by centrifugation (1800 g, 10 min, 4 1C), and

washed with and resuspended in 1 mL of glucose-free

minimal medium, and the fluorescence intensities of mito-

chondria were then visualized with an LSM 510 META laser

scanning confocal microscope (Zeiss, Oberkochen, Ger-

many; lexcitation = 543 nm, lemission = 615 nm), using a long-

pass filter.

Susceptibility to oxidative stress-generating andantifungal agents

MIC values for the oxidants tBOOH, H2O2, menadione

sodium bisulfite [MSB; a soluble superoxide-generating

agent (Pocsi et al., 2005)] and NaOCl were determined

by a serial dilution method performed in SDB medium

under standard yeast propagation conditions. The tested

concentration ranges were: 0–32 mmol L�1 for tBOOH;

0–250 mmol L�1 for H2O2; 0–5 mmol L�1 for MSB; and

0–100 mmol L�1 for NaOCl. The lowest oxidant concentra-

tions that inhibited the growth of C. albicans completely

were regarded as MICs. Because oxidative stress may sig-

nificantly change the antimycotic susceptibility of C. albi-

cans (Gyetvai et al., 2007), MIC values were also determined

for fluconazole, 5-fluorocytosine and amphotericin B in

RPMI-1640 and SDB media according to the National

Committee for Clinical Laboratory Standards guidelines for

antifungal susceptibility testing (Song et al., 2003; Gyetvai

et al., 2006).

Elements of antioxidative defense, andproduction of ROS

The specific activities of a series of antioxidant enzymes, as

well as the intracellular glutathione (GSH) and glutathione

disulfide (GSSG) concentrations, were determined in both

C. albicans AF06 and C. albicans ATCC 14053 (Pocsi et al.,

2004).

Both C. albicans AF06 and C. albicans ATCC 14053 cells

cultured in 20-mL aliquots of SDB medium for 12 h were

exposed to 0, 1 and 6 mmol L�1 (C. albicans AF06) or 0 and

1 mmol L�1 (C. albicans ATCC 14053) tBOOH for 5 h,

harvested by centrifugation (1800 g, 10 min, 4 1C), washed

three times with 0.1 mol L�1 potassium phosphate buffer

(pH 7.0), and resuspended in 10 mL of potassium phosphate

buffer. The cell-free extracts were prepared by

X-pressing and centrifugation (Emri et al., 1999). The

specific GR, glutathione peroxidase (GPx), glutathione-S-

transferase (GST), g-glutamyltranspeptidase (gGT), cata-

lase, SOD and glucose-6-phosphate dehydrogenase (G6PD)

activities were measured in the supernatants of the cell-free

extracts as described elsewhere (Emri et al., 1997, 1999). The

protein contents of the cell-free extracts were measured by a

modification of the Lowry method (Peterson, 1983).

For GSH and GSSG determinations, C. albicans cells were

cultured and exposed to tBOOH in 100-mL aliquots of SDB

in shake flasks (volume = 500 mL). tBOOH-treated C. albi-

cans cells were separated from 50-mL aliquots of the cultures

by centrifugation (1800 g, 10 min, 4 1C) and were washed

with ice-cold distilled water. Candida albicans cells were

resuspended in 4 mL of ice-cold 5% (w/v) 5-sulfosalicylic

acid by vigorous mixing, and were left at 0 1C

for 20 min (Emri et al., 1999). After centrifugation



(11 000 g, 10 min, 4 1C), the supernatants were neutralized

with triethanolamine at 0 1C, and the specific intracellular

GSH and GSSG concentrations were determined according

to Anderson (1985). The protein contents were determined

from the other 50-mL aliquots of the cultures in these

experiments.

Accumulation of intracellular peroxide and superoxide

was always detected by the formation of 20,70-dichlorofluor-

escin (DCF) from 20,70-dichlorofluorescin diacetate and

ethidium from dihydroethidium, respectively (Emri et al.,

1997; Gyervai et al., 2006).

Fatty acid composition and lipid peroxidationproducts

Oxidative stress tolerance in C. albicans cells was also related

to changes in the concentrations of saturated and unsatu-

rated fatty acids and lipid peroxidation products (Pocsi

et al., 2004).

In fatty acid composition analyses, the culture volumes

were 100 mL, and the cultivations were carried out in 500-

mL shake flasks for 17 h. Total lipid fractions were extracted

from 2-mL aliquots of X-pressed, 1 mmol L�1 (C. albicans

ATCC 14053) or 6 mmol L�1 (C. albicans AF06) tBOOH-

treated and untreated control cell suspensions with 3 mL of

chloroform/methanol 2 : 1, and then with 2 mL of chloro-

form according to the method of Bligh & Dryer (1959). The

organic fractions were separated and evaporated to dryness

under N2. Fatty acids were analyzed after HCl-catalyzed

methylation in methanol, using a Hewlett Packard 5890 gas

chromatograph coupled to a Hewlett Packard 5970 mass

spectrometer. Neutral lipids were identified with thin-layer

chromatography as described elsewhere (Varga et al., 1997).

For determination of lipid peroxidation products, thio-

barbituric acid-reactive substances (TBARS) in 1 mmol L�1

(C. albicans ATCC 14053) or 6 mmol L�1 (C. albicans AF06)

tBOOH-treated and untreated C. albicans cultures were

detected as described elsewhere (Balla et al., 1991; Gyetvai

et al., 2007). The concentration of TBARS was always

estimated using a standard curve of malondialdehyde pre-

pared by acid hydrolysis of malondialdehyde tetrabutylam-

monium salt (Balla et al., 1991; Gyetvai et al., 2007).

Conjugated dienes were also measured in the supernatants

spectrophotometrically according to Balla et al. (1991).

Respiration

Elements of the respiratory chain are likely to elicit and/or

maintain lipid peroxide chain reactions in the presence of

O2 (Evans et al., 1998) and, hence, the tBOOH tolerance of

the AF06 mutant may also originate from a reduction in

cytochrome c-dependent respiration. Alternatively, the in-

duction of salicylhydroxamate (SHAM)-resistant respira-

tion may also confer oxidative stress tolerance to the AF06

mutant (Huh & Kang, 2001). In respiration measurements,

C. albicans AF06 and ATCC 14053 cultures (volu-

me = 100 mL; SDB) were treated with 0, 1 and 6 mmol L�1

tBOOH for 5 h, and the respiration of the cultures was

measured in a built-in-house oxygraphic cell (volu-

me = 15 mL) at 28 1C using an OXY 320-type oxygen

electrode (WTW, Weilheim, Gemany) (Bahr & Bonner,

1973; Emri et al., 2004). To inhibit the cytochrome

c-dependent pathway or the alternative oxidase (AOX) path

way, 2.5 mmol L�1 KCN and 0.750 mmol L�1 SHAM were

used, respectively. The contribution of the AOX pathway to

total respiration was estimated by measuring both the KCN-

resistant and the SHAM-sensitive parts of respiration.

Statistics and chemicals

Unless otherwise indicated, means and SDs calculated from

four independent experiments are presented. The variations

between experiments were estimated by SDs, and the

statistical significance of changes in physiologic parameters

was estimated with Student’s t-test. Only probability levels

of P � 5% were regarded as being indicative of statistical

significance.

Unless otherwise indicated, all the chemicals were pur-

chased from the Sigma-Aldrich Ltd, Budapest, Hungary.

Results

The development of tBOOH tolerance resultedin decreased hypha-forming capability inC. albicans

A series of C. albicans mutants with increased tBOOH

tolerance was developed by continuous cultivation of

C. albicans ATCC14053 in the presence of increasing con-

centrations of tBOOH (2–16 mmol L�1). MICtBOOH values

and specific GR activities increased steadily as a function of

the tBOOH concentrations employed, reaching their max-

ima when C. albicans cells were exposed to 8 mmol L�1

tBOOH (Fig. 1). At higher tBOOH concentrations, e.g.

16 mmol L�1, progressive cell death was observed, character-

ized by sharply declining cell vitality and biomass. tBOOH-

tolerant mutants always developed under continuous culti-

vation with tBOOH, and the acquired tBOOH tolerance was

heritable, as demonstrated by unaltered MICs and specific

GR activities even after 10 subsequent passages of the

C. albicans AF01–10 mutants on tBOOH-free SDA plates.

There was no difference between the colony sizes of the

AF01–10 mutants and the parental ATCC14053 strain in 2-,

3- and 5-day spread-plate cultures, and no colony with a

petite morphology was observed. Unlike the parental

C. albicans ATCC 14053 strain, the AF01–10 mutants

showed negligible hypha formation on Spider agar (Fig. 2);

that is, the development of oxidative stress tolerance



coincided with the reduction in hypha formation in the

tBOOH-tolerant strains.

Increased tBOOH tolerance altered theproduction of virulence factors and moderatedpathogenicity but did not affect antigenicity

Among the virulence attributes studied, extracellular aspar-

tic protease production of the tBOOH-tolerant C. albicans

AF06 mutant increased, while extracellular phospholipase

production decreased significantly in comparison to the

control parental strain (Table 1). In addition to the lack of

hypha formation on Spider agar, germ tube formation in

sheep serum and pseudohypha formation on corn-meal agar

were also reduced considerably. On the other hand, no

difference in the chlamydospore-forming capabilities of the

mutant and the parental strains was recorded on corn-meal

agar (Table 1).

Human PMNLs recognized opsonized mutant and par-

ental control C. albicans cells equally, as demonstrated by the

dose-dependent but quite similar superoxide production by

PMNLs recoded at different yeast/PMNL cell ratios

(1 : 100–1 : 25; Fig. 3).

The LD50 values determined for mice for the parental and

the mutant C. albicans strains were 8� 104 and 40� 104

cells, respectively; that is, the tBOOH-tolerant C. albicans

AF06 mutant showed markedly decreased pathogenicity

when injected intravenously. It is worth noting that the yeast

cells isolated from infected mice were all identified as

C. albicans, and that their MICtBOOH values and specific

GR activities were identical to those of C. albicans AF06.

When C. albicans cells were injected intraperitoneally, the

numbers in the peritoneal cavities were identical for both

the mutant and the parental control strains, and both

GR

[m

kat

(kg

prot

ein)

–1]

tBOOH concentration (mmol L–1)

00

4

8

12

16

20(a)

(b)

2 4 8 16

tBOOH concentration (mmol L–1)

00

1

2

3

4

5

2 4 8 16

MIC

tBO

OH

(mm

ol L

–1)

Fig. 1. Changes in tBOOH tolerance (a) and specific GR activity (b) of

Candida albicans ATCC14053 exposed to stepwise increasing concen-

trations (0–8 mmol L�1) of tBOOH. Mutant generation and characteriza-

tion were repeated 10 times in independent experiments. Neither

MICtBOOH values nor specific GR activities are shown for 16 mmol L�1

tBOOH treatments, because of the progressive cell death observed in the

cultures.

Fig. 2. Comparison of the colony morphologies of Candida albicans

ATCC14053 (a) and AF06 (b) after 10 days of incubation on Spider agar

at 28 1C. The AF06 strain had smooth colony borders, indicating that it

was impaired in yeast ! hyphae morphologic transitions. Similar colony

morphologies with no (four other mutants) or negligible (five mutants)

hypha formation were observed with the other Candida albicans tBOOH-

tolerant strains selected. Bar = 5 mm.



colonized the kidneys with hyphal cell morphology after

24 h (Fig. 4).

tBOOH tolerance did not affect cell size andmorphology

The average cell sizes of the mutant and parental strains

determined by scanning electron microscopy were the same:

19� 11mm3 (n = 28) and 19� 8 mm3 (n = 36), respectively.

No differences could be seen on the cell surfaces either

(data not shown). Similarly, no alterations in cellular

Table 1. Comparison of the in vitro virulence attributes of Candida albicans ATCC 14053 and Candida albicans AF06

Virulence attributes C. albicans ATCC 14053 C. albicans AF06

Germ tube formation in sheep serum (%)

30 min 43� 4 6�3�

60 min 78� 8 59�2�

180 min 95� 7 97�5

Secreted aspartic protease activity [A280 nm� 1010 (cell number)�1] 8.4� 0.5 16.6�1.7�

Secreted phospholipase activityw 1.0� 0.1 0.24�0.01�

Hypha formation on solid Spider medium 1 �Pseudohypha formation on corn-meal agar

48 h 1 �62 h 1 1

Chlamydospore formation on corn-meal agar 1 1

All data represent means� SD calculated from four independent experiments.�Significant (P � 5%) differences between the control and the tBOOH-tolerant strains. P-values were calculated using Student’s t-test.wPhospholipase activities were characterized with the (1/Pz)�1 values, where Pz stands for the phospholipase zone calculated by dividing the colony

diameter by the cloudy-zone-plus-colony diameter measured after 48 h of incubation at 37 1C Fekete-Forgacs et al. (2000); Gyetvai et al. (2007).

0

5

10

15

20

25

1:25 1:501:100Candida : PMNL ratio

Supe

roxi

de a

nion

pro

duct

ion

[nm

ol (

106

cells

)–1 h

–1 ]

Fig. 3. Comparison of dose-dependent superoxide production by poly-

morphonuclear leukocytes (PMNLs) in contact with opsonized Candida

albicans ATCC14053 (gray columns) and Candida albicans AF06 (black

columns) cells added in ratios of 1 : 100 to 1 : 25 (Candida/PMNLs).

Fig. 4. Kidney cortex sections interwoven with Candida albicans hyphae

as visualized by the periodic acid Schiff technique. Mice were injected

intraperitoneally with 106 Candida albicans ATCC14053 cells (a) ort-

BOOB-tolerant Candida albicans AF06 cells (b). Mice were sacrificed 24 h

after infection. Bar = 25 mm.



ultrastructure, with regard to the cell wall, nuclei and

mitochondria, were found in transmission electron micro-

graphs of the AF06 and ATCC 14053 strains (Fig. 5).

Staining with the mitochondrion-selective MitoTracker Red

probe also indicated that mitochondria of both C. albicans

strains had identical size and morphology as well as correct

organelle functioning when the cells were grown on a

glucose carbon source (data not shown).

tBOOH tolerance also increased tolerance to aseries of oxidative stress-generating agents andantimycotics

As shown in Table 2, the fourfold increase in the tBOOH

tolerance of the AF06 mutant in comparison to the parental

strain coincided with twofold increases in the H2O2, MSB

and NaOCl tolerances. Importantly, the enhanced oxidative

stress tolerance also resulted in a significantly increased

tolerance to the antifungal drugs fluconazole, amphotericin

B, voriconazole, and 5-fluorocytosine (Table 2).

The tBOOH tolerance-related physiologicchanges shed light on the continuous inductionof the antioxidative defense system

Candida albicans AF06 possessed 1.5–4 times higher specific

GR, G6PD, GPx, catalase and SOD activities than its

parental strain, clearly indicating continuous induction of

the antioxidative defense system of the mutant even in

tBOOH-free culture medium (Table 3). When C. albicans

ATCC14053 was challenged with 1 mmol L�1 tBOOH, the

specific activities of GR and GPx increased significantly,

whereas the same treatment did not increase further the

high antioxidant enzyme activities typical of the tBOOH-

tolerant mutant (data not shown). On the other hand, the

specific GR, G6PD, GPx, catalase and SOD activities were

also responsive to tBOOH-triggered oxidative stress in the

C. albicans AF06 mutant when the tBOOH concentration

was increased to 6 mmol L�1. Interestingly, neither GST nor

gGT, enzymes of the GSH-dependent detoxification path-

way (Pocsi et al., 2004), were responsive to oxidative stress

or showed significant changes in activity in either the

parental or the tBOOH-tolerant mutant strains (Table 3).

The C. albicans AF06 mutant accumulated more GSH and

much more GSSG than its ancestor, resulting in an overall

negative change in the GSH/GSSG redox balance (Table 3).

Candida albicans ATCC14053 cells accumulated ROS

(superoxide, peroxide) as well as GSSG in high concentra-

tions, with a 1.65-fold concomitant overproduction of GSH

(Table 3), when challenged by 1 mmol L�1 tBOOH. The

C. albicans AF06 strain produced more peroxide under

unstressed conditions than its parental strain, but no over-

production of peroxide and GSH was recorded in the

mutant cells challenged with 6 mmol L�1 tBOOH, whereas

intracellular superoxide levels increased more than 10-fold

[0.17–1.8 nmol ethidium (mg protein)�1; Table 3]. Again,

1 mmol L�1 tBOOH did not affect GSH and ROS metabo-

lism of the tBOOH-tolerant mutant (data not shown).

N

MCw(a)

(b)

Cw

N

M

1µm

1µm

Fig. 5. Transmission electron microscopy images of the cellular ultra-

structures of Candida albicans ATCC14053 (a) and Candida albicans

AF06 (b). Cw, M and N stand for cell wall, mitochondrion and nucleus,

respectively. Note that both strains possessed mitochondria of typical size

and morphology and with cristae.

Table 2. Comparison of the tolerances of Candida albicans ATCC

14053 and Candida albicans AF06 against oxidative stress-generating

and antifungal agents

C. albicans

ATCC 14053

C. albicans

AF06

Oxidative stress-generating agents

tBOOH [MIC (mmol L�1)] 4 16

H2O2 [MIC (mmol L�1)] 32 64

MSB [MIC (mmol L�1)] 0.6 1.2

NaOCl [MIC (mmol L�1)] 0.01 0.02

Antifungal agents

Fluconazole [MIC (mg mL�1)] 0.5 2

Voriconazole [MIC (mg mL�1)] 0.016 0.032

Amphotericin B [MIC (mg mL�1)] 0.5 1

5-Fluorocytosine [MIC (mg mL�1)] 0.125 0.25

tBOOH, t-butylhydroperoxide; MSB, menadione sodium bisulfite.



Altered lipid composition indicated significantlipid peroxidation in unstressed C. albicansAF06 cells

As far as the lipid compositions of the C. albicans AF06

mutant and the C. albicans ATCC14053 parental strains are

concerned, no significant differences in the phospholipids,

either in quantities or in phospholipid patterns, were found

between the two strains (data not shown), whereas signifi-

cant differences in neutral lipids were detected. The total

amount of neutral lipids was significantly higher in the

tBOOH-tolerant mutant than in the parental strain

[610� 130 vs. 530� 120 mg (g protein)�1]. The neutral

lipid fraction of the mutant contained significantly reduced

quantities of ergosterol as well as free fatty acids, and was

composed of significantly elevated amounts of unidentified

sterols and diacyl glycerols in comparison to that of the

parental strain (Fig. 6a). There was no significant difference

in the triacyl glycerol contents of the C. albicans cells

(Fig. 6a).

The fatty acid composition of total lipid extracts of

C. albicans AF06 and ATCC14053 was also analyzed, and

there was no significant difference in the total amount of

fatty acids between the two strains; however, several differ-

ences in the amounts of individual fatty acids were found

(Fig. 6b). The saturated fatty acid (SFA) content of the

mutant was significantly higher, whereas the monounsatu-

rated and polyunsaturated fatty acid (MUFA and PUFA)

contents were significantly lower, than those recorded in the

parental strain (Fig. 6b). These changes were attributed to

elevated levels of palmitic and strearic acids, and reduced

levels of palmitoleic, oleic, linoleic and linolenic acids, in the

tBOOH-resistant strain (data not shown).

After exposure to 1 mmol L�1 (C. albicans ATCC 14053)

or 6 mmol L�1 (C. albicans AF06) tBOOH, unidentified

sterol and PUFA concentrations decreased, whereas diacyl

Table 3. Comparison of selected physiologic parameters of Candida albicans ATCC 14053 and Candida albicans AF06 in the absence and presence of

t-butylhydroperoxide (tBOOH)

Physiologic parameters

C. albicans

ATCC 14053

C. albicans ATCC

1405311 mmol L�1

tBOOH

C. albicans

AF06

C. albicans

AF0616 mmol L�1

tBOOH

Specific antioxidant enzyme activities

G6PD [mkat (kg protein)�1] 6� 1 8�2 10�0.7� 12.9�0.8��

GPx [mkat (kg protein)�1] 0.055� 0.004 0.17�0.02� 0.2�0.01� 0.33�0.04��

GR [mkat (kg protein)�1] 1.1� 0.15 2�0.1� 2.9�0.3� 3.4�0.2��

GST [mkat (kg protein)�1] 0.094� 0.008 0.11�0.02 0.09�0.01 0.1�0.02

gGT [mkat (kg protein)�1] 0.026� 0.004 0.022�0.005 0.029�0.004 0.028�0.003

Catalase [kat (kg protein)�1] 1.2� 0.3 1.5�0.3 1.8�0.2� 2.4�0.2��

SOD [unit (mg protein)�1] 3.7� 0.8 4.5�0.6 5.9�0.3� 11�3��

Stress-related metabolites

Peroxide [nmol DCF (mg protein)�1] 0.040� 0.08 0.060�0.010� 0.056�0.012� 0.049�0.010

Superoxide [nmol ehtidium (mg protein)�1] 0.2� 0.03 0.28�0.06� 0.17�0.03 1.8�0.3��

GSH [nmol (mg protein)�1] 158� 11 260�17� 208�11� 211�12�

GSSG [nmol (mg protein)�1] 0.5� 0.03 2.5�0.1� 1.9�0.06� 2.6�0.1��

GSH/GSSG 319� 21 105�9� 109�9� 82�8��

Lipid peroxidation products

Lipid hydroperoxide [nmol (mg protein)�1] 3.2� 0.2 3.9�0.4� 3.6�0.3� 10�1��

Conjugated dienes [A353 nm (mg protein)�1] 0.04� 0.01 0.09�0.03� 0.08�0.02� 0.22�0.02��

TBARS [nmol (mg protein)�1] 0.009� 0.003 Not determined 0.042�0.017� Not determined

Respiration

Total respiration [nmol s�1 (mg protein)�1] 32� 6 40�9 33�6 36�7

Cytochrome c-dependent respiration

[nmol s�1 (mg protein)�1]

11� 2 16�3� 5�1� 4�1�

Alternative oxidase-dependent respiration

[nmol s�1 (mg protein)�1]

21� 4 24�5 28�5� 32�6�

Cytochrome c-dependent/alternative respiration 0.52� 0.14 0.66�0.19 0.18�0.05� 0.13�0.04�

All data represent means� SD calculated from four independent experiments.�Significant (P � 5%) differences between the tBOOH-treated control and untreated tBOOH-tolerant strains in comparison to those of the untreated

control strain.��Significant (P � 5%) differences between the tBOOH-treated and untreated Candida albicans AF06 cells.

P-values were calculated with Student’s t-test.

G6PD, glucose-6-phosphate dehydrogenase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GST, glutathione-S-

transferase; GSSG, glutathione disulfide; gGT, g-glutamyltranspeptidase; TBARS, thiobarbituric acid-reactive substances; DCF, 2 0,70-dichlorofluorescein.



glycerol concentrations increased, in both the AF06 mutant

and ATCC14053 parental strains (Fig. 6a and b). tBOOH-

generated oxidative stress also increased the MUFA content

of the mutant, but lowered the ergosterol and elevated the

free fatty acid contents of the parental strain (Fig. 6).

Peroxidation of unsaturated fatty acids was more promi-

nent in C. albicans AF06, because the amounts of all lipid

peroxidation products measured were significantly higher in

the mutant than in the parental strain (Table 3). Treatment

with tBOOH increased the conjugated diene and lipid

hydroperoxide contents of the cells in both strains (Table 3).

The increased conjugated diene and TBARS production

observable in unstressed C. albicans AF06 cells, together

with the increased SFA and decreased MUFA and PUFA

contents (Table 3; Fig. 6), were indicative of progressive lipid

peroxidation.

The tBOOH-tolerant AF06 mutant showeddecreased cyanide-sensitive respiration andincreased alternative respiration

The tBOOH-tolerant mutant had significantly lower cya-

nide-sensitive cytochrome c-dependent respiration than was

found in the parental strain (55% reduction; Table 3).

However, this difference was not reflected in total respira-

tion, owing to the high cyanide-resistant alternative respira-

tion detectable in the mutant strain (33% increase; Table 3).

tBOOH treatments did not increase the total respiration

markedly, but stimulated cyanide-sensitive respiration in

the parental strain (Table 3). Moreover, effective channeling

of electrons towards cyanide-resistant alternative respiration

was demonstrated by the remarkably low ratios of cyanide-

sensitive respiration/cyanide-resistant respiration in C. albi-

cans AF06 cultures either treated or not treated with

tBOOH. It is important to note that KCN-resistant respira-

tion and SHAM-sensitive respiration were always equal, and

the remaining respiration measured in the presence of both

KCN and SHAM was always o 10% of total respiration in

each C. albicans culture tested.

Discussion

Oxidative stress-tolerant C. albicans mutants developed

when C. albicans ATCC 14053 cells were exposed to increas-

ing concentrations of tBOOH (Fig. 1; Table 2), and the

acquired tBOOH tolerance was heritable for at least 10

passages on tBOOH-free SDA plates. In addition, the

tBOOH-tolerant C. albicans AF06 mutant presented iden-

tical MICtBOOH values and specific GR activities even after 2

weeks following intravenous injection into mice (data not

shown). The AF06 strain also had increased tolerance to the

major oxidants produced by phagocytes, including H2O2,

superoxide and OCl�, as demonstrated by the twofold

increases in the MICH2O2, MICMSB and MICNaOCl values,

respectively (Table 2). As hypothesized, the oxidative stress-

tolerant C. albicans AF01–10 mutants possessed significantly

reduced hypha-forming capability, and the AF06 mutant

was also characterized by a reduction in germ tube and

pseudohypha formation (Table 1; Fig. 2).

Morphologic transitions are inevitably needed for suc-

cessful tissue and organ invasion and dissemination within

the host, and are therefore important virulence factors

(Braun & Johnson, 1997; Lo et al., 1997; Gow et al., 2002;

Mavor et al., 2005). Considering other virulence attributes,

the AF06 mutant produced less extracellular phospholipase,

*

* **

***

*

0

10

20

30

40

50

60(a)

(b)

***

**

*

*

*

*

*

**

SFA MUFA PUFA

erg erg-e FFA DG TG

Dis

trib

utio

n of

neu

tral

lipi

ds (

mol

%)

0

10

20

30

40

50

70

60

Dis

trib

utio

n of

fat

ty a

cids

(m

ol %

)

Fig. 6. Distribution of neutral lipids (a) and fatty acids (b) in the Candida

albicans ATCC14053 strain (control; white and gray columns) and the

tBOOB-tolerant Candida albicans AF06 strain (dark gray and black

columns) in the absence (first columns) and presence of tBOOH

(tBOOH = 1 mmol L�1 for the control strain, and tBOOH = 6 mmol L�1 for

the tBOOB-tolerant strain; second columns). erg, ergosterol; erg-e,

unidentified sterols; FFA, free fatty acids; DAG, diacyl glycerols; TAG,

triacyl glycerols; SFA, saturated fatty acids; MUFA, monounsaturated

fatty acids; PUFA, polyunsaturated fatty acids. Columns and bars

represent means� SD calculated from four independent experiments.�Significant (P � 5%) differences between the lipid contents of tBOOH-

treated control and untreated tBOOH-tolerant strains in comparison to

those of the untreated control strain. ��Significant (P � 5%) differences

between the lipid contents of tBOOH-treated and untreated Candida

albicans AF06 cells. P-values were calculated with Student’s t-test.



which is highly expressed in yeast cells and pseudohyphae,

and effectively degrades cell membrane components (Ibra-

him et al., 1995; Leidich et al., 1998), but its aspartic

protease production (required for tissue invasion) (Calder-

one & Fonzi, 2001) exceeded that of C. albicans ATCC 14053

(Table 1). PMNLs recognized both the mutant and the

parental strain equally (Fig. 3), indicating that the mutation

leading to the oxidative stress-tolerant phenotype of C.

albicans AF06 did not affect antigenicity. The reduced

capability of the fungus to undergo dimorphic morphologic

transitions, together with decreased phospholipase produc-

tion, resulted in a less pathogenic strain with a fivefold

higher LD50 value in mice when compared to that of the

ATCC 14053 strain (40� 104 vs. 8� 104 cells). Therefore,

the development of oxidative stress tolerance, which is

thought to be advantageous for C. albicans when it interacts

with immune system cells (Mavor et al., 2005), seems to be

disadvantageous when the fungus escapes from blood vessels

and invades deep organs. Our results are in accordance with

previous observations that exposure of C. albicans to

PMNLs and macrophages in whole blood (Fradin et al.,

2003, 2005; Lorenz et al., 2004) hindered the production of

some important virulence attributes, including dimorphic

switches (Table 1) (Fradin et al., 2003).

Because these findings illuminate some new features of

C. albicans–host interactions that may lead to improved

therapeutic approaches to combat candidiasis, e.g. via the

development of drugs or drug combinations that initiate

and maintain oxidative stress in C. albicans cells (Gyetvai

et al., 2007), a wide spectrum of physiologic studies was

performed to shed light on the molecular background of

heritable oxidative stress tolerance in this pathogen.

In terms of cell physiology, C. albicans AF06 possessed

high specific GR, G6PD, GPx, catalase and SOD activities,

which, unlike in the parental strain, did not respond to

1 mmol L�1 tBOOH treatment but were still inducible by

6 mmol L�1 tBOOH (Table 3). The persistently high antiox-

idant enzyme activities coincided with high endogenous

oxidant (peroxide, GSSG, lipid hydroperoxide) levels and

a GSH/GSSG redox imbalance (Table 3). Progressive lipid

peroxidation was also demonstrated in the mutant by

increased conjugated diene and TBARS production and by

the increased SFA and decreased MUFA and PUFA contents

(Table 3; Fig. 6).

The physiologic changes typical of the C. albicans AF06

mutant (continuous redox imbalance, high peroxide level,

accelerated lipid peroxidation) are highly suitable for the

promotion and maintenance of continuous induction of

the antioxidative defense system, as presented in Table 3.

The intracellular accumulation of ROS and GSH/GSSG

redox imbalance have been shown to govern the expression

of large groups of genes in a fungal genome (Pocsi et al.,

2005), and linoleic acid hydroperoxide (a lipid hydroper-

oxide) was also shown to trigger genome-wide transcrip-

tional changes in Sa. cerevisiae (Alic et al., 2004).

Furthermore, the lipid peroxidation intermediate malon-

dialdehyde (a TBARS component) elicited an adaptive stress

response in baker’s yeast (Turton et al., 1997; Evans et al.,

1998).

The origin of peroxide and lipid peroxidation products

observable in high concentrations in unstressed C. albicans

AF06 cultures (Table 3) has yet to be studied, but the

imbalance between cyanide-sensitive respiration and

SHAM-sensitive respiration (Table 3) may be indicative of

heritable mtDNA damage resulting in increased electron

leakage from the electron transport chain (Wei et al., 1998;

Osiewacz & Borghouts, 2000; Wei & Lee, 2002; Genova et al.,

2004; Doudican et al., 2005). Because elements of the

respiratory chain contribute to the initiation and mainte-

nance of lipid peroxide chain reactions, any reduction in

cytochrome c-dependent respiration, as seen in the C.

albicans AF06 mutant, provides benefits in coping with lipid

peroxidation-triggered oxidative stress (Evans et al., 1998).

Importantly, the growth rate and biomass production of C.

albicans AF06 on a glucose carbon source were comparable

to those of the parental strain (data not shown), and

C. albicans AF06 also possessed large cristate mitochondria

(Fig. 5) that functioned correctly (MitoTracker Red probe;

data not shown). In these respects, C. albicans AF06

resembled the respiratory mutant C. albicans KRD-8 gener-

ated by acriflavine treatment, which retained 20% of the

respiration rate of the parental strain (Aoki & Ito-Kuwa,

1987; Ito-Kuwa et al., 1988).

Although the in vivo selection of C. albicans mutants with

increased antioxidative potential but with concomitantly

decreased virulence seems unlikely, some more recent find-

ings by other research groups may challenge this view. For

example, blockage of respiration by respiratory chain in-

hibitors induced decreased sensitivity to azoles in C. glabra-

ta (Brun et al., 2003), which may allow the selection of less

pathogenic respiratory mutants of this species in azole-

treated patients (Brun et al., 2005). Moreover, exposure to

antimicrobial peptides such as salivary histatins may also

select C. albicans respiratory mutants (Gyurko et al., 2000).

This means that the in vivo selection of oxidative stress-

tolerant respiratory C. albicans mutants with decreased

pathogenicity cannot be excluded when C. albicans cells

have to adapt to environmental stress caused by concurrent

attacks of immune system cells and antimycotics. It is

important to note that the oxidative stress-tolerant C.

albicans AF06 strain, which did not completely lose its

hypha-forming ability and virulence in mice (Fig. 4), also

showed twofold to fourfold increased tolerance to a series of

frequently used antimycotics (fluconazole, voriconazole,

amphotericin B, 5-fluorocytosine; Table 2). The important

questions of exactly how cross-tolerance against oxidants



and antimycotics develops, and how the combinations of

oxidants and antimycotics disturb the physiology of C.

albicans cells, e.g. through altering membrane composition

and fluidity (Fig. 6) (Gyetvai et al., 2006, 2007) and/or

synergistically enhancing oxidative stress (Sokol-Anderson

et al., 1986; Liu et al., 2005; Gyetvai et al., 2007), will be

addressed in a more detailed future study.

Acknowledgements

We are grateful to Dr L. Majoros, University of Debrecen, for

providing us with the C. albicans ATCC 14053 strain. We

thank Dr L. Csernoch, Mrs B. Dienes, Mrs E. Falusi, MrsE.

Nagy and Mr I. Pocsi, University of Debrecen, and Ms K.

Takacs, University of Pecs, for their valuable help in plan-

ning and performing the experimental work. This work was

supported financially by grants OTKA T 34157, OTKA T

34315 OTKA T 62092, NKFP 3/50/2001 and RET-06/2004.

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Documents

Development of oxidative stress tolerance resulted in reduced ability to undergo morphologic transitions and decreased pathogenicity in a t-butylhydroperoxide-tolerant mutant of Candida