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1 Área de Microbiología, Facultad de Biología, Universidad de Murcia, E-30071 Murcia, Spain
2 Departamento de Microbiología y Ecología, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain
3 Departamento de Bioquímica y Biología Molecular B e Inmunología, Universidad de Murcia, E-30071 Murcia, Spain
Correspondence
Juan-Carlos Argüelles
arguelle{at}um.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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/atc1 mutant showed that it was unable to grow on exogenous trehalose as sole carbon source. Unlike actively growing cells from the parental strain (CAI4), the atc1 null mutant displayed higher resistance to environmental insults, such as heat shock (42 °C) or saline exposure (0.5 M NaCl), and to both mild and severe oxidative stress (5 and 50 mM H2O2), which are relevant during in vivo infections. Parallel measurements of intracellular trehalose and trehalose-metabolizing enzymes revealed that significant amounts of the disaccharide were stored in response to thermal and oxidative challenge in the two cell types. The antioxidant activities of catalase and glutathione reductase were triggered by moderate oxidative exposure (5 mM H2O2), whereas superoxide dismutase was inhibited dramatically by H2O2, where a more marked decrease was observed in atc1 cells. In turn, the atc1 mutant exhibited a decreased capacity of hypha and pseudohypha formation tested in different media. Finally, the homozygous null mutant in a mouse model of systemic candidiasis displayed strongly reduced pathogenicity compared with parental or heterozygous strains. These results suggest not only a novel role for the ATC1 gene in dimorphism and infectivity, but also that an interconnection between stress resistance, dimorphic conversion and virulence in C. albicans may be reconsidered. They also support the hypothesis that Atc1p is not involved in the physiological hydrolysis of endogenous trehalose.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Berman & Sudbery, 2002; Akins, 2005; Chauhan et al., 2006). Superficial candidiasis is common in skin, oral cavity, gastrointestinal tract and vagina in otherwise healthy individuals (Verduyn Lunel et al., 1999; Calderone & Fonzi, 2001; Eggimann et al., 2003). However, C. albicans also causes life-threatening septicaemic infections in debilitated patients (Patterson, 2005; Chauhan et al., 2006). Furthermore, the incidence of nosocomial bloodstream candidiasis has increased dramatically in recent years (McNeil et al., 2001; Patterson, 2005). Together with some well-characterized virulence factors, the low selective toxicity of currently available antifungal therapies and the increase in resistant strains might account for this high prevalence (Akins, 2005; Mukherjee et al., 2005).
Some specific physiological features of C. albicans have been invoked as contributory factors of pathogenicity (Odds, 1994; Calderone & Fonzi, 2001; Gow et al., 2002). C. albicans is a dimorphic organism capable of switching from yeast to mycelial (hypha and pseudohypha) morphology. Although both forms are present in infected hosts (Cannon et al., 1994; Gow et al., 2002; Sudbery et al., 2004), a predominance of filamentous structures is apparently required for effective tissue invasion (Lo et al., 1997). During the progress of an in vivo infection, the microbial pathogens should combat or adapt to the stresses imposed by the host defence machinery (Vázquez-Torres & Balish, 1997; Chauhan et al., 2006). Following ingestion by active phagocytes, for instance, C. albicans encounters high levels of oxidants that must be counteracted. In fact, this fungus is relatively resistant to oxidative stress and has evolved a proficient antioxidant response to survive after phagocytosis (Vázquez-Torres & Balish, 1997; Chauhan et al., 2006).
In the search for novel antifungal targets, the non-reducing disaccharide trehalose has emerged as a very promising candidate, as this sugar is present in bacteria, fungi and plants, but not in mammals (Argüelles, 2000; Elbein et al., 2003). Trehalose acts mainly as an energy source and a stress protector in yeast and fungi (Thevelein, 1996; Argüelles, 2000; Elbein et al., 2003). Trehalose metabolism has been investigated intensively in C. albicans in connection with fungal dimorphism, oxidative stress and protection, as has its possible involvement in virulence. In fact, the genes implicated in the biosynthesis of this disaccharide, namely TPS1 (trehalose-6-phosphate synthase) and TPS2 (trehalose-6-phosphate phosphatase), have been cloned and characterized, and the corresponding homozygous null mutants have been constructed. Disruption of TPS1 impairs hypha formation and decreases infectivity (Zaragoza et al., 1998), whereas TPS2 is involved in cell integrity and pathogenicity, but is not required for dimorphic transition (Van Dijck et al., 2002; Zaragoza et al., 2002). Furthermore, the role of trehalose-catabolism enzymes remains unclear. The NTC1 gene, encoding the neutral trehalase, lacks a specific function during dimorphic transition and pathogenicity (Eck et al., 1997). In turn, trehalose appears to play a main protective role, specifically against oxidative stress rather than other environmental challenges (Alvarez-Peral et al., 2002).
We have recently cloned the ATC1 gene, which encodes a cell wall-linked acid trehalase (Pedreño et al., 2004) in C. albicans. The atc1 null mutant was unable to grow on trehalose as sole carbon source, confirming that Atc1p is required to hydrolyse exogenous trehalose (Pedreño et al., 2004). In the present paper, we used this atc1/atc1 mutant to analyse the putative changes in trehalose metabolism (synthesis, hydrolysis and endogenous content) in response to different stress challenges during the development of hyphal structures, and to test the hypothetical implication of the ATC1 gene in the infectivity of this fungus. Our results suggest a new role for Atc1p in hypha formation and virulence, but they dismiss the interest of trehalose as a potentially useful antifungal target.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. A detailed description of the constructions and procedures followed to obtain this set of mutants is given elsewhere (Pedreño et al., 2004). A new strain, CEY-1 (CAI4-URA3), was constructed. The vector CIp10 (Murad et al., 2000) was utilized to integrate the URA3 gene into the genome of CAI4; to target the integration of CIp10, it was digested with NcoI, which cuts uniquely within the RP10 sequence. Digested plasmid was used to transform C. albicans CAI4 according to Gietz et al. (1995). Transformed cells were selected as Ura+ in minimal medium without uridine; three independent positive transformants were examined and the correct integration of the URA3 marker was confirmed by Southern blot.
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, and on Spider medium plates. Germ-tube formation was monitored by means of a phase-contrast light microscope with a haemocytometer. When required, clumped cells were dispersed prior to microscopic examination by mild sonication (10–15 s). At least 200 cells were counted each time and the percentage of dimorphism was represented as the ratio of germ tube-forming cells to the total number of cells.
Heat-shock, oxidative and osmotic stress treatments.
Cultures were grown in YPD until the exponential phase (OD600=0.8–1.0) and were then divided into several identical aliquots, which were treated with different H2O2 concentrations (5–50 mM), 0.5 M NaCl or 42 °C for oxidative, osmotic or heat-shock treatments, respectively, or maintained without treatment as a control and incubated at 28 °C for 1 h.
Viability was determined after samples had been diluted appropriately with sterile water by plating in triplicate on solid YPD after incubation for 2–3 days at 28 °C. Between 30 and 300 colonies were counted per plate. Survival was normalized to control samples (100 % viability).
Preparation of cell-free extracts.
After exposure to different stresses, samples from the cultures were harvested and resuspended at known densities (10–15 mg ml–1, wet weight) in MES extraction buffer (100 mM), pH 6.0, containing 5 mM cysteine and 0.1 mM PMSF. The cellular suspensions were transferred into small, pre-cooled tubes (1.0 cm diameter) with 1.5 g Ballotini glass beads (0.45 mm diameter). Cells were broken by vibrating the tubes vigorously in a vortex mixer. The tubes were cooled quickly on ice. The crude extract was then centrifuged at 10 000 g for 5 min and the pellet was resuspended in the same buffer at the initial density. For antioxidant assays, the supernatant fraction was filtered through Sephadex G-25 NAP columns (Amersham Biosciences), previously equilibrated with 50 mM sodium phosphate buffer, pH 7.8, to remove low-molecular-mass compounds.
Enzymic assays.
The assay for acid trehalase was performed by incubating 50 µl cell-wall pellet with 200 µl trehalose (200 mM) prepared in 200 mM sodium citrate, pH 4.5, containing 2 mM EDTA. The reaction for neutral trehalase activity contained 50 µl cell-free extract (25–100 µg protein) and 200 µl trehalose (200 mM) prepared in 25 mM MES, pH 7.1, 125 µM CaCl2. The reactions were incubated at 30 °C for 30 min and stopped by heating in a water bath at 100 °C for 5 min. The amount of glucose released was determined by using the glucose oxidase–peroxidase method. Specific activity is expressed as nmol glucose min–1 (mg protein)–1. Trehalose-6-phosphate synthase activity was measured at 40 °C in the supernatants of cell-free extracts as described previously (Argüelles et al., 1999). Specific activity is expressed as nmol trehalose min–1 (mg protein)–1.
Catalase activity was determined at 240 nm by monitoring the removal of H2O2, as described elsewhere (González-Párraga et al., 2003). Glutathione reductase (GR) activity was assayed by measuring the glutathione disulfide (GSSG)-dependent oxidation of NADPH after Hernández et al. (1999). Measurements of superoxide dismutase (SOD) were carried out spectrophotochemically by the ferricytochrome c method using xanthine/xanthine oxidase as the source of 
radicals (González-Párraga et al., 2003). Data of enzymic activity were normalized in relation to a control measurement (100 %).
Other measurements.
Intracellular trehalose was measured by following the method described by Blázquez et al. (1994). Briefly, cell samples (20–50 mg, wet weight) were washed, resuspended in 2 ml water and boiled for 30 min with occasional shaking. The concentration of trehalose released in the supernatant was determined with commercial trehalase (Sigma). The assay contained 90 µl 25 mM sodium acetate buffer, pH 5.6, 100 µl cell-free supernatant and 10 µl trehalase (2 units ml–1). After incubation overnight at 37 °C, the amount of glucose produced was estimated by the glucose oxidase–peroxidase procedure. Parallel controls were run to correct the basal glucose levels.
Growth was monitored by measuring the OD600 of cultures in a Shimadzu UV spectrophotometer. Protein was estimated by the Lowry method, with BSA as standard.
Virulence in mice.
Overnight yeast cultures were refreshed for 4 h at 28 °C in YPD to an OD600 of 0.8. Groups of eight female SWISS CD-1 mice (6–8 weeks old), weighing 21–25 g each, were inoculated in the lateral caudal vein with 1.0x106 viable C. albicans cells suspended in 150 µl PBS. Survival was monitored daily over a 1 month period. Survival curves were calculated according to the Kaplan–Meier method by using the PRISM program (GraphPad Software) and compared by using the log-rank test. A P value of 
0.05 was considered significant.
Statistical analysis.
All experiments were repeated at least three times, with the exception of the viability analysis and the infectivity assay in mice, which were performed twice with consistent results. Differences between the values recorded were tested for significance in accordance with Duncan's multiple range test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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/atc1). Fig. 1 shows that CAI4 performed as the most sensitive strain under all environmental conditions tested. In the case of oxidative treatments, exposure to a moderate concentration of H2O2 (5 mM) resulted in a higher percentage of survival in the mutant after 1 h incubation (an almost 3-fold increase) than in parental cells, whilst a high concentration of H2O2 (50 mM) was responsible for a dramatic loss of viability in both strains. Again, ATC2-2 sustained a better resistance to oxidative stress (Fig. 1).
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). In contrast, a heat shock at 42 °C for 1 h triggered an approximately equivalent rate of cell death in both strains (approx. 25 %; Fig. 1b). When the treatment was prolonged to 3 h, however, the atc1/atc1 cells exhibited a greater capacity to withstand heat shock than CAI4, whereas the sensitivity against saline and oxidative stress remained practically identical (Fig. 1c).
Changes in trehalose metabolism and trehalose content during stress treatments
The same cultures submitted to several stress conditions were used to monitor simultaneously possible changes in the enzymic activities involved in both trehalose metabolism and the intracellular content of this disaccharide. Acute oxidative stress (50 mM H2O2) promoted a significant synthesis of intracellular trehalose in both strains, whilst low H2O2 levels (5 mM) induced only a weak increase in disaccharide storage (Table 2). This increase in trehalose appears to be due to the partial inhibition of neutral trehalase after the addition of 5 mM H2O2 and the concomitant activation of trehalose synthase (Table 3). On the other hand, it is likely that the marked accumulation of sugar in response to 50 mM H2O2 exposure is due to a dramatic inhibition of Ntc1p (80 %), as Tps1p activity also underwent a degree of inactivation (around 20 %; Table 3). As expected, acid trehalase was virtually undetectable in the mutant (Table 3). A notable fact is that this activity followed the same trend in response to oxidative stress as that recorded for neutral trehalase (Table 3). However, Atc1p makes no significant contribution to the endogenous content of trehalose (Alvarez-Peral & Argüelles, 2000; Pedreño et al., 2004).
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) in the two trehalase activities, with the exception of acid trehalase in the mutant, where this enzyme is not functional (Table 3). Interestingly, trehalose synthase activity was inactivated in both ATC2-2 and CAI4 cells, albeit to a lower extent in the latter strain (20 % decrease; Table 3). This could account for the lack of variability of the endogenous trehalose content upon saline stress in relation either to the control assay in CAI4 cells or to the slight decrease in the mutant (Table 2).
A pronounced increase (6-fold) of intracellular trehalose in both parental and mutant cultures was observed when a heat shock at 42 °C was applied to exponential-phase cells (Table 2). This net synthesis arises from a 2-fold activation of trehalose synthase activity compared with the control assay (Table 3). The heat shock also triggered an increase in neutral trehalase activity (Table 3). The apparently contradictory parallel rise in endogenous trehalose and its hydrolase Ntc1p has been explained convincingly as the need of trehalose mobilization to yield enough energy to ensure proper cellular recovery after heat shock (Singer & Lindquist, 1998).
Induction of antioxidant activities in response to H2O2 treatments
The metabolic study was completed by analysing the hypothetical changes recorded in a set of activities that play an antioxidant role, such as catalase, GR and total SOD. For this purpose, exponential-phase cultures of the ATC2-2 and CAI4 strains were subjected to identical H2O2 exposures. The presence of a non-lethal H2O2 concentration (5 mM) promoted a clear rise in catalase activity (almost 40 %) in parental cells after incubation for 1 h; however, the increase was more conspicuous (2-fold) in the ATC2-2 cells (Fig. 2a). In both cell types, treatment with a potentially lethal H2O2 concentration (50 mM) caused a partial decrease in catalase activity in relation to the values recorded in control assays (Fig. 2a). As regards GR, the results shown in Fig. 2(b) support the fact that this activity was strikingly high when either mild or acute oxidative exposure (5 or 50 mM H2O2) was applied for 1 h (Fig. 2b). These data are consistent with the oxidative stress-induced GR activation observed in a tps1 mutant deficient in trehalose synthesis (González-Párraga et al., 2003).
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). Measurements of total SOD activity after incubation with 5 mM H2O2 revealed a 40 % decrease of the initial activity in CAI4 cells (Fig. 2c) and a slightly higher loss of activity in mutant cells (Fig. 2c). Likewise, toxic H2O2 concentrations (50 mM) were harmful for this enzymic activity, which suffered a larger reduction in ATC2-2 cultures (80 %) than in parental-type cells (60 %; Fig. 2c).
Effect of ATC1 gene disruption on dimorphic conversion
The relationship between hypha formation and trehalose metabolism has been disputed in the past (Ram et al., 1984; Argüelles et al., 1999). Genetic evidence demonstrated conclusively that neither the TPS1 (trehalose-6-phosphate synthase) nor the NTC1 (neutral trehalase) gene is involved in this process (Eck et al., 1997; Zaragoza et al., 1998). In order to analyse the hypothetical role of the ATC1 gene in dimorphism, exponential-phase cultures from the ATC1-2 (ATC1/atc1), ATC2-2 (atc1/atc1) and CAI4 strains were subjected to a set of conditions under which efficient hyphal growth may be induced, e.g. Spider medium (Fig. 3) or human serum at 37 °C (Fig. 4a), whereas stationary-phase cells were induced in Lee's medium, as reported by Elorza et al. (1985) (Fig. 4b). All of these procedures caused a considerable delay in the degree of germ-tube formation in the ATC2-2 null mutant compared with its isogenic parent CAI4 (Figs 3 and 4). The heterozygous ATC1-2 mutant exhibited an intermediate percentage of filamentous growth in Spider and Lee's media (Figs 3 and 4b), although it was similar to the value measured in CAI4 cells in YPD supplemented with human serum (Fig. 4a). When equivalent experiments were performed with the corresponding URA+ instead of URA– strains, using CEY-1 strain as control, the results obtained revealed that the homozygous atc1 mutant (ATC2-1) was also defective in germ-tube formation (data not shown).
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). ATC2-2 cultures, which exhibited a lower capacity to produce germ tubes (Fig. 4), accumulated more endogenous trehalose than CAI4 cells (Table 4). Hence, there is a clear lack of correlation between the degree of hyphal formation and the concentration of stored trehalose, which supports the hypothesis that disaccharide is not required to enter dimorphic transition (Zaragoza et al., 1998; Argüelles et al., 1999).
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), the corresponding URA+ transformants were obtained from the strains used in this study. In order to determine whether deletion of ATC1 affects the virulence of C. albicans, groups of selected mice were injected in the lateral caudal vein with 1x106 cells of the following strains: CEY-1 (CAI4-URA+), the ATC1-1 (ATC1/atc1 URA+) and ATC2-1 (atc1/atc1 URA+) mutants, as well as the reconstituted strain with the intact ATC1 gene (ATC2-3). The pattern of mouse survival is shown in Fig. 5. After 16 days, no survivors were found in the group of mice inoculated with parental cells, whereas >75 % of those inoculated with the ATC2-1 mutant were still alive after at least 1 month (Fig. 5). Mice infected with the heterozygous mutant (ATC1-1) showed an intermediate behaviour between mice infected with the CEY-1 cells and those infected with the homozygous mutant (Fig. 5), whereas injection of the ATC2-3 strain (with the reintroduced ATC1 gene) evidenced a behaviour similar to that recorded in parentally and heterozygously infected mice (Fig. 5). The experiment was repeated once more with consistent results. These data support the idea that at least one ATC1 copy is required to regain infectivity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Elbein et al., 2003); (ii) trehalose accumulation may be a specific protective mechanism of pathogenic fungi during in vivo infection (Alvarez-Peral & Argüelles, 2000); (iii) the genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) are determinants of virulence, and the corresponding tps1 and tps2 null mutants show high sensitivity to severe oxidative stress (Zaragoza et al., 1998, 2002; Alvarez-Peral et al., 2002; Van Dijck et al., 2002). The design of antifungals against the cell wall has received priority, and the echinocandins, which act as potent inhibitors of glucan synthases, are currently in clinical use (Denning, 2003).
The cloning of the ATC1 gene (Pedreño et al., 2004) has led to it being considered as a promising candidate for the development of antifungal compounds, as its product, acid trehalase, combines both features, namely Atc1p is linked to the cell wall and it is required for the hydrolysis of exogenous trehalose (Pedreño et al., 2004). To explore this possibility, we performed a phenotypic analysis of the atc1 null mutant to study its resistance to several stresses, its ability to enter dimorphism and its degree of virulence. Thus, atc1/atc1 cells showed a greater capacity to withstand oxidative and osmotic challenges than those of its parental strain (Fig. 1). Of the antioxidant enzymes monitored, only catalase and GR appear to act as cell protectors against oxidative exposure, whereas SOD was inhibited (Fig. 2). As Atc1p is a cell wall-linked protein, the stress resistance in atc1 cells is presumably due to structural modifications associated with ATC1 disruption, as the endogenous trehalose content accumulated in mutant and parental cultures was similar (Table 2).
The need for trehalose mobilization as a putative factor involved in dimorphic transition and the hydrolytic role played by acid trehalase in this process have been disputed (Ram et al., 1984; Zaragoza et al., 1998; Argüelles et al., 1999). Available data support the hypothesis that human serum-induced dimorphism is independent of trehalose hydrolysis (Argüelles et al., 1999), although no conclusive genetic evidence exists. For this reason, we analysed the ability of the ATC2-2 (atc1) null mutant to enter dimorphic conversion in response to various stimuli. The morphogenetic programme that induces hyphal outgrowth underwent a slight delay in growing atc1 blastoconidia (Figs 3–5). This phenotype of retardation in dimorphism might, once again, be the consequence of modifications in the cell-wall structure after the ATC1 double disruption, rather than to changes in stored trehalose. However, a direct effect on the signalling pathways governing cell morphology cannot be ruled out (Berman & Sudbery, 2002).
In comparison to the parental and heterozygous strains, the homozygous atc1 mutant showed a significant reduction in pathogenicity during long-term infection in mice (Fig. 5). This attenuated virulence is clearly comparable with similar phenotypes caused by the double disruption of the TPS1 and TPS2 genes involved in trehalose biosynthesis (Zaragoza et al., 1998, 2002; Van Dijck et al., 2002). However, our data discount the possibility that this lower infectivity could be due to differences in trehalose accumulation; whereas the tps1 and tps2 null mutants exhibited a negligible or clearly diminished reduction of endogenous trehalose (Zaragoza et al., 1998, 2002; Van Dijck et al., 2002), the atc1 cells strongly accumulated disaccharide upon exposure to several stresses (Table 2). Therefore, we conclude preliminarily that the expectations raised concerning the potential usefulness of the ATC1 gene as an antifungal target are ill-founded, because atc1 cells simultaneously displayed reduced filamentation and virulence, but enhanced resistance to heat shock and oxidative stress.
Why is the atc1 mutant more resistant to stress in vitro, but less virulent in vivo? The success of an opportunistic pathogen during infection stems, in part, from its capacity to counteract the defence reactions from the host, including the high levels of reactive oxygen species (ROS) released after ingestion by phagocytes (Vázquez-Torres & Balish, 1997). The atc1 cells might withstand the damage provoked by endogenous ROS, as they store a large amount of intracellular trehalose when incubated at 37 °C with human serum (Table 4) and the disaccharide in C. albicans acts as a specific protectant against oxidative stress (Alvarez-Peral et al., 2002). On the other hand, it is well known that structural perturbations of cell-wall integrity affect the capacity of host recognition, adhesion and further infection of host cells (Gow et al., 2002; Bates et al., 2006). Furthermore, recent evidence has demonstrated the critical importance of N-glycans embedded in the outer layer of the cell wall in host–fungal interaction and virulence (Bates et al., 2006), and that Atc1p contains 20 potential N-glycosylation sites (Pedreño et al., 2004). Hence, disruption of ATC1 probably alters the external surface stability, with a subsequent reduction in virulence.
The connection between hypha formation and virulence in C. albicans is controversial (Cannon et al., 1994; Kobayashi & Cutler, 1998; Gow et al., 2002). In general, mycelium shape appears to be associated with infectivity, and Lo et al. (1997) demonstrated how mutants unable to filament are avirulent. However, all cellular types are present in clinical lesions caused by other dimorphic fungi (Histoplasma, Blastomyces), where the yeast-like form is predominant in pathogenesis (Cannon et al., 1994). With regard to the virulence of mutants altered in trehalose metabolism, the link between dimorphic conversion and virulence is unclear. Thus, whilst disruption of the TPS1 gene impairs the yeast-to-hypha transition and decreases infectivity (Zaragoza et al., 1998), TPS2 is apparently necessary for proper cell integrity (Van Dijck et al., 2002; Zaragoza et al., 2002). However, the homozygous mutant showed strongly reduced virulence, without affecting the capacity to produce germ tubes and subsequent hypha development (Van Dijck et al., 2002; Zaragoza et al., 2002). The performance of the atc1 mutant is quite similar to that of tps1, although the atc1 cells synthesize a high quantity of intracellular trehalose during supply of human serum at 37 °C (Tables 2 and 4). Indeed, such a level of trehalose might be relevant in the course of an in vivo infection.
On the other hand, the NTC1 gene, which encodes the cytosolic neutral trehalase, does not seem to play a significant role in growth and infectivity (Eck et al., 1997) or to participate in the protective response against oxidative stress (Pedreño et al., 2006). Hence, ATC1 is the first gene involved in trehalose catabolism that is necessary for virulence in C. albicans.
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ACKNOWLEDGEMENTS |
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Edited by: J. F. Ernst
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 30 October 2006;
revised 16 January 2007;
accepted 17 January 2007.
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