Area de Microbiologia, Facultad de Biologia, Universidad de Murcia, Campus de Espinardo, E-30071 Murcia, Spain1
Instituto de Investigaciones Biomédicas del CSIC, Unidad de Bioquimica y Genética de Levaduras, 28029 Madrid, Spain2
Author for correspondence: Juan-Carlos Argüelles. Tel: +34 968 36 71 31. Fax: +34 968 36 39 63. e-mail: arguelle{at}um.es
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ABSTRACT |
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Keywords: TPS1, cell protector, oxidative stress, adaptive response, opportunistic yeast pathogen
Abbreviations: ROS, reactive oxygen species; T-6P synthase, trehalose-6-phosphate synthase
a Present address: Albert Einstein College of Medicine, Microbiology and Immunology. Golding Building, room 701, 1300 Morris Park Avenue, Bronx, NY 10461, USA
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INTRODUCTION |
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Yeast cells represent a very convenient model for studying cellular responses to oxidants and other physiological stresses. In this lower eukaryote, accumulation of the non-reducing disaccharide trehalose has been convincingly demonstrated as one of the main defence mechanisms against different stress conditions, such as heat shock, nutrient starvation, dehydration, toxic chemicals and oxidative stress (Wiemken, 1990 ; Thevelein, 1996
; Estruch, 2000
; Argüelles, 2000
). Trehalose seems to act by stabilizing membranes and native proteins as well as by suppressing the aggregation of denatured proteins (Singer & Lindquist, 1998
). The expression of genes encoding trehalose-6-phosphate synthase (TPS1) and neutral trehalase (NTH1) is induced in response to specific stress challenges (Nwaka et al., 1995
; Nwaka & Holzer, 1998
; Zähringer et al., 1997
, 2000
).
In this study, we investigate the role of trehalose as a specific cellular protector against oxidative stress in the opportunistic yeast pathogen Candida albicans, a ubiquitous human commensal in healthy people. However, among the immunocompromised population, C. albicans behaves as a highly virulent pathogen and invasive candidiasis is frequent in AIDS patients, transplant recipients, neonates and those undergoing cancer or antibiotic therapy (Cutler, 1991 ; Coleman et al., 1993
; Odds, 1988
, 1994
). In addition, C. albicans is a dimorphic fungus that can grow either as budding yeast cells (blastoconidia) or as mycelium (hypha and/or pseudohypha) (Shepherd et al., 1985
; Odds, 1988
; Cutler, 1991
; Molero et al., 1998
; Brown & Gow, 1999
). Morphological transition is a contributory factor in pathogenesis, the mycelial phase being predominant during host tissue colonization. However, many underlying signals that govern morphogenesis remain to be elucidated (Lo et al., 1997
; Brown & Gow, 1999
; Ernst, 2000
). Previous studies on trehalose metabolism in C. albicans indicate that neither its accumulation in blastoconidia nor its further hydrolysis is essential for the yeast-to-hypha dimorphic conversion (Zaragoza et al., 1998
; Argüelles et al., 1999
). However, trehalose storage confers thermotolerance on exponentially growing cells (Argüelles, 1997
). According to the results presented here, the disaccharide protects cells against drastic oxidative stress, but is not required for the adaptive oxidative stress response, a process that might be relevant in the course of an in vivo infection.
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METHODS |
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Yeast cell cultures were grown at 28 °C by shaking in a medium consisting of 2% peptone, 1% yeast extract and 2% galactose (YPgal). The strains were maintained by periodic subculturing on solid YPgal. Escherichia coli DH5 transformation and recombinant DNA manipulation followed standard procedures (Sambrook et al., 1989
).
Oxidative stress treatments and acquired oxidative stress tolerance.
Cultures were grown in YPgal until the exponential phase (OD600=0·81·3) and then divided into several identical aliquots, which were treated with different H2O2 concentrations (or maintained without H2O2 as a control) and incubated at 28 °C for 1 h. For experiments on acquired oxidative tolerance or cross-tolerance, a given sample was incubated with 0·5 mM H2O2 or at 37 °C for 1 h and immediately challenged with 50 mM H2O2. Viability was determined after appropriate dilution of the samples with sterile water by plating in triplicate on solid YPgal. Between 30 and 300 colonies were counted per plate. Survival was normalized to control samples (100% viability).
Preparation of permeabilized cells and cell-free extracts.
At the indicated times, aliquots were harvested, washed and resuspended at known densities (usually 1015 mg ml-1, wet weight) in 10 mM MES buffer pH 6·0. For the measurement of acid trehalase, these samples were treated with 10% (v/v) of a mixture composed of toluene/ethanol/Triton X-100 (TET; 1:4:0·2 by vol.). The suspension was shaken in a vortex for 5 min at 4 °C, washed and resuspended at initial density in 10 mM MES pH 6·0.
Neutral trehalase and trehalose-6-phosphate synthase (T-6P synthase) activities of cell-free extracts were determined as described previously (Argüelles et al., 1999 ), except that no CaCl2 was included in the extraction buffer (10 mM MES, pH 6·0).
Enzymic assays.
Acid trehalase was assayed by incubating 50 µl permeabilized cells (0·51·0 mg wet weight) with 200 µl 200 mM trehalose prepared in 100 mM sodium acetate pH 5·6. The assay for neutral trehalase contained 50 µl cell-free extract (25100 µg protein) and 200 µl 200 mM trehalose prepared in 25 mM MES pH 7·1, 125 µM CaCl2. The reactions were incubated at 37 °C for 1530 min and stopped by heating in a water bath at 100 °C for 5 min. The glucose released was determined using the glucose oxidase-peroxidase method. One unit of trehalase is defined as the amount of enzyme that hydrolyses 1 µmol trehalose (2 µmol glucose) per min. Specific activity is expressed either as mU (mg wet weight)-1 (external trehalase) or as mU (mg protein)-1 (neutral trehalase).
T-6P synthase was measured at 40 °C in the supernatants of cell-free extracts as described by Argüelles (1997) . Specific activity is expressed as mU (mg protein)-1.
Other determinations.
Intracellular trehalose was extracted from 2050 mg yeast samples in 2 ml boiling water and the concentration measured with commercial trehalase (Sigma) following the method described by Blázquez et al. (1994) , except that glucose was estimated by the glucose oxidase-peroxidase procedure. Parallel controls were run to correct for the basal levels of glucose.
Growth was monitored by measuring the OD600 of cultures or by direct cell counting with a haemocytometer; at least 200 colonies were counted for each determination. Protein was estimated by the Lowry method with BSA as standard.
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RESULTS |
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Whether there is a correlation between the resistance of CAI.4 cells and their endogenous trehalose content has also been determined. The oxidative challenge promoted a significant rise in trehalose in CAI.4 cells (Table 1), but failed to induce any trehalose increase in the tps1/tps1 mutant counterpart (Table 1
). However, survival of both strains upon H2O2 exposure did not exactly mirror parallel changes in the intracellular trehalose content (Fig. 1b
, Table 1
). The trehalose accumulation in CAI.4 cultures was the result of the T-6P synthase activation (TPS1p) and the concomitant inactivation of neutral trehalase (NTH1p), the latter being the enzyme responsible for trehalose mobilization in response to different physiological signals (Thevelein, 1996
; Zähringer et al., 1997
; Argüelles, 2000
) (Table 1
). The presence of cycloheximide had a slight inhibitory effect on the oscillations recorded in both enzymic activities (results not shown). Overexpression of the TPS1 gene suppressed the susceptibility of tps1/tps1 cells to oxidative stress treatments (Fig. 1b
) and restored intracellular trehalose levels (Table 2
). These data provide additional evidence that CaTPS1p is the sole activity involved in trehalose biosynthesis in C. albicans (Zaragoza et al., 1998
).
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Cross-tolerance has been demonstrated previously in S. cerevisiae, where tolerance to one type of stress confers cross-protection to another stress (Lewis et al., 1995 , 1997
). Therefore, we investigated whether thermotolerance-inducing treatments, which have always been associated with trehalose storage (Hottiger et al., 1987
; Thevelein, 1996
), would confer oxidative protection in C. albicans. As has been demonstrated for other C. albicans strains (Argüelles, 1997
), exponential CAI.4 and tps1/tps1 cultures incubated at 28 °C underwent a dramatic viability reduction when submitted to a severe heat stress (52·5 °C for 5 min). Preincubation of equivalent samples at standard human body temperature (37 °C) led to a substantial increase of cells from both strains able to withstand the further heat shock (three- to fivefold for CAI.4 and two- to threefold for tps1/tps1, respectively, data not shown). By contrast, basal level of trehalose in CAI.4 growing cultures at 28 °C was very low and not increased by mild heat exposure at 37 °C, while trehalose was virtually nil in the tps1/tps1 mutant (Table 3
). Remarkably, trehalose synthesis in C. albicans seems to be markedly temperature-dependent, since no trehalose accumulation was observed upon incubation at 37 °C (Zaragoza et al., 1998
; Table 3
), although a further upshift to 42 °C clearly promoted the carbohydrate storage (Argüelles, 1997
; Zaragoza et al., 1998
).
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DISCUSSION |
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Heat shock elicits in yeast a complex response that involves the coordinated action of Hsp104, Hsp70 and trehalose accumulation (Estruch, 2000 ). By contrast, protection against moderate or high osmolarity mainly depends on the high osmolarity glycerol (HOG) pathway, which controls the osmotic induction of glycerol-synthesizing enzymes (Brewster et al., 1993
). As regards oxidative stress, the capacity to withstand the deleterious effect of ROS requires both enzymic (catalase or superoxide dismutase) and non-enzymic (gluthatione and thioredoxin) components (Jamieson, 1998
; Estruch, 2000
).
Data obtained here support the hypothesis that trehalose is needed as protectant for growing C. albicans blastoconidia directly submitted to a severe oxidative treatment (higher than 10 mM H2O2) (Figs 1 and 2
). The H2O2-sensitive phenotype shown by tps1/tps1 cells was efficiently suppressed by overexpression of the TPS1 gene (Figs 1
and 2
). However, direct exposure to oxidants promotes a lower degree of trehalose accumulation than other stress conditions. In a previous study (Jamieson et al., 1996
), trehalose synthase was not considered as a specific antioxidant enzyme. The storage of the disaccharide, however, appears to be largely dispensable during the adaptive response triggered by a previous incubation with a low non-lethal concentration of H2O2, the degree of tolerance being roughly equivalent in both wild-type and tps1/tps1 cultures (Fig. 2
, Table 2
). Furthermore, although C. albicans displays a cross-tolerance protective mechanism that increases the resistance to H2O2 induced by mild heat exposure (37 °C), this occurs with no specific change in trehalose metabolism (Fig. 3
, Table 3
).
Therefore, present knowledge suggests that although trehalose must be considered one of the principal protective factors against ROS, other elements must be involved in the response to oxidative stress. Exposure to moderate stress seems to activate an alternative pathway, which would build up the resistance, even when a further severe stress is applied. However, when non-adapted cells are subjected to an intense stress, the ability to synthesize trehalose appears to be essential. Consequently, we propose that the response to ROS involves two different steps: a first step would be triggered immediately by the direct stress and requires the intracellular accumulation of trehalose, while the second would need some time before being fully operative, and be dependent on (a) pathway(s) as yet unidentified. In the absence of trehalose synthesis, this second mechanism would become operative after a previous mild exposure to a non-lethal stress.
The study of oxidative stress responses in C. albicans may have important clinical repercussions in assessing the progress of in vivo infections and the respiratory defensive mechanism of phagocytes. Thus, the formation of ROS and other oxidants by phagocytes plays an essential function in combating fungal infections (Murphy, 1991 ) and the effective antifungal effect of azole drugs on ergosterol biosynthesis is in part due to the sensitization of C. albicans to the reactive oxygen-dependent microbicidal system produced by macrophages (Shimokawa & Nakayama, 1992
). In this context, our preliminary results suggest that the tps1/tps1 mutant displays greater sensitivity to the phagocytosis brought about by mouse macrophages than its counterpart CAI.4 (results not shown), which might explain the low rate of infectivity exhibited by tps1/tps1 cells when inoculated in mice (Zaragoza et al., 1998
).
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ACKNOWLEDGEMENTS |
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Received 26 March 2002;
accepted 11 April 2002.