Abstract

In the pathogenic yeast Candida albicans, the MAP-kinase Hog1 mediates an essential protective role against oxidative stress, a feature shared with the transcription factor Cap1. We analysed the adaptive oxidative response of strains with both elements altered. Pretreatment with gentle doses of oxidants or thermal upshifts (28→37 and 37→42 °C) improved survival in the face of high concentrations of oxidants (50 mM H2O2 or 40 mM menadione), pointing to a functional cross-protective mechanism in the mutants. The oxidative challenge promoted a marked intracellular synthesis of trehalose, although hog1 (but not cap1) cells always displayed high basal trehalose levels. Hydrogen peroxide (H2O2) induced mRNA expression of the trehalose biosynthetic genes (TPS1 and TPS2) in the tested strains. Furthermore, oxidative stress also triggered a differential activation of various antioxidant activities, whose intensity was greater after HOG1 and CAP1 deletion. The pattern of activity was dependent on the oxidant dosage applied: low concentrations of H2O2 (0.5–5 mM) clearly induced catalase and glutathione reductase (GR), whereas drastic H2O2 exposure (50 mM) increased Mn-superoxide dismutase (SOD) isozyme-mediated SOD activity. These results firmly support the existence in C. albicans of both Hog1- and Cap1-independent mechanisms against oxidative stress.

Introduction

The clinical incidence of opportunistic mycosis has increased dramatically in the last two decades to become a significant public health problem (Chauhan, 2006; Pfaller & Diekema, 2007). A worldwide increase in the immunocompromised and ageing population, the limited arsenal of efficient and selective antifungals and the growing number of fungi that show resistance to conventional antibiotics are just some of the reasons for this worrying scenario (Eggimann, 2003; Akins, 2005; Chauhan, 2006). Although several clinical isolates of Aspergillus, Crypotococcus (alongside several dimorphic fungi) and some ‘non-albicans’ species of Candida are frequently identified as being responsible for nosocomial outbreaks, Candida albicans is still the most prevalent infectious fungus in humans, where it is a common inhabitant of the oral cavity and the gastrointestinal and genitourinary tracts. In healthy individuals, the capacity of this opportunistic pathogen to proliferate and colonize deep tissues and vital organs is rigorously precluded by both the innate and the acquired immunological mechanisms. However, immunodeficiency, extensive surgery and chemotherapy, together with certain diseases and physiological perturbations, give rise to septicaemic candidiasis, which can become life-threatening in the absence of antifungal chemotherapy (Eggimann, 2003; Pfaller & Diekema, 2007).

A compelling body of evidence collated in recent years points to an essential role for the HOG pathway in the physiology of C. albicans. The main conclusions are summarized as follows: (1) Hog1 is involved in the response to oxidative and osmotic stresses as well as resistance to heavy metals, (2) Hog1 is involved in cell wall biosynthesis, (3) a functional Hog1 represses the yeast to hypha dimorphic transition and controls chlamydospore formation and (4) the hog1 mutant displays reduced virulence in mice and enhanced susceptibility to phagocytic cells. Closely related to its role in oxidative stress, a control for Hog1 has been proposed recently in the respiratory metabolism of C. albicans (San José, 1996; Alonso-Monge, 1999, 2003, 2009; Smith, 2004).

Nevertheless, Hog1 does not appear to be the only signalling pathway involved in the cell defence against oxidative stress. The cell integrity pathway, mediated by Mkc1, is also activated upon exposure to certain oxidants, although Mkc1 phosphorylation is partially Hog1 dependent (Navarro-García, 2005). The C. albicans bZIP transcription factor CAP1 (Alarco & Raymond, 1999) also participates in the resistance to oxidants through a mechanism unrelated to the Hog1 cascade (Alonso-Monge, 1999, 2003; Smith, 2004). In addition, the nonreducing disaccharide trehalose appears to play a specific protective role against acute hydrogen peroxide (H2O2) and heat-shock treatments, rather than against other stresses (Alvarez-Peral, 2002). How these distinct pathways are interconnected and the hypothetical existence of a general oxidative signalling regulator remain an open question. Through an analysis of the oxidative stress-induced responses in hog1 and cap1 mutants, we present data supporting the existence of several, apparently unrelated, but cooperative, protective mechanisms in C. albicans.

Materials and methods

Yeast strains and growth conditions

RM100 (ura3Δ∷imm434/ura3Δ∷imm434 his1Δ∷hisG/his1Δ∷hisG-URA3-hisG) was used as the parental strain. A detailed description of the constructions and procedures to obtain the set of homozygous hog1 and cap1 isogenic derivative mutants used throughout this study is reported elsewhere (Alonso-Monge, 2003).

Unless otherwise stated, yeast cell cultures were grown at 37 °C with shaking in a medium consisting of 2% peptone, 1% yeast extract and 2% glucose (YPD). Strains were maintained at 4 °C by periodic subculturing in solid YPD. Usually, preinoculated overnight cultures were harvested, resuspended in fresh YPD and incubated further until they reached the exponential phase. The growth was monitored turbidimetrically by measuring the OD600 nm of cultures or by direct cell counting using a haemocytometer; at least 200 cells were counted for each determination. Protein was estimated using the Lowry (1951) method with bovine serum albumin as the standard.

Oxidative stress treatments and acquired oxidative stress tolerance

Cultures were grown in YPD until the exponential phase (OD600 nm=0.8–1.0) and then divided into several identical aliquots, which were treated with different H2O2 and menadione concentrations (or maintained without oxidants as a control) and incubated at 37 °C for 1 h. For experiments on acquired oxidative tolerance, a given sample was incubated with H2O2 or menadione (0.5 mM) at 37 °C for 1 h and immediately challenged with 50 mM H2O2 or 40 mM menadione. For ‘cross-tolerance’ assays, logarithmic growing cells were transferred either from 28 to 37 °C or from 37 to 42 °C for 1 h, challenged with 50 mM H2O2 and then incubated at the initial temperature.

Viability was determined after appropriate dilution of the samples with sterile water by triplicate plating on solid YPD or by spotting on solid YPD. Survival was normalized to control samples (100% viability).

Protein extracts and immunoblot analysis

The RM100 strain was grown overnight at 28 or 37 °C. Then, both cultures were refreshed and incubated at the same temperature until they reached an OD of 1. The flasks were shifted to 37 or 42 °C, respectively, and samples were taken at 0, 10, 40 and 60 min after the shift. Cell extracts were obtained as indicated previously (Martin, 2000). Protein concentration was assessed from A280 nm and equal amounts of proteins were loaded onto gels. Blots were probed with phospho-p44/42 MAP kinase (Thr202/Tyr204) (E10) (Ab-p42-44P; Cell Signaling Technology Inc.), ScHog1 polyclonal antibody (Ab-ScHog1; Santa Cruz Biotechnology Inc) and Ab-p-p38 (Thr180/Tyr182)-R (Anti–p38-P; Santa Cruz Biotechnology Inc.) and developed according to the manufacturer's conditions using the Hybond ECL kit (Amersham-Pharmacia Biotech AB).

Preparation of cell-free extracts

Samples subjected to different stress exposures were harvested at the indicated times, washed with cold water and resuspended at known densities (10–15 mg mL−1, wet weight) in 100 mM 4-morpholine-ethanesulphonic acid (MES), pH 6.0, containing 5 mM cysteine and 0.1 mM PMSF. The cellular suspensions were transferred into small precooled tubes (1.0 cm diameter) with 1.5 g Ballotini glass beads (0.45 mm diameter). The cells were broken by vigorous vibration of the tubes in a vortex mixer at six cycles of 45 s each. The crude extract was then centrifuged at 10 000 g for 5 min and the supernatant fraction obtained was filtered through Sephadex G-25 NAP columns (Amersham-Pharmacia Biotech AB) equilibrated previously with 50 mM K-phosphate buffer, pH 7.8, in order to remove low-molecular-weight compounds that might interfere with enzymatic activities, especially superoxide dismutase (SOD).

Assay of enzymatic antioxidant activities

Catalase activity was determined at 240 nm by monitoring the removal of H2O2, and glutathione reductase (GR) activity was assayed by measuring the GSSG-dependent oxidation of NADPH following previously published procedures, as described by González-Párraga (2003). Measurements of SOD activity were carried out spectrophotochemically by the ferricytochrome c method using xanthine/xanthine oxidase as the source of O2 radicals (McCord & Fridovich, 1969); 1 U of activity was defined as the amount of enzyme necessary to produce a 50% inhibition of the ferricytochrome c reduction rate. All the determinations were repeated at least three times. Differences between the values recorded were tested for significance according to Duncan's multiple-range test.

Extraction and determination of intracellular trehalose

Intracellular trehalose was measured following the procedure described elsewhere (Pedreño, 2007). 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 into the supernatant was determined with commercial trehalase (Sigma). The assay contained 90 μL 25 mM sodium acetate buffer, pH 5.6, 100 μL of cell-free supernatant and 10 μL trehalase (2 U mL−1). After an overnight incubation at 37 °C, the glucose produced was estimated using the glucose oxidase–peroxidase procedure. Parallel controls were run to correct the basal glucose levels.

Real-time quantitative RT-PCR (qRT–PCR) analysis

Total RNA was isolated from exponential yeast cultures exposed to oxidative stress for 0, 15, 30 and 60 min using the ‘mechanical disruption’ protocol and RNeasy mini kits with column DNAse treatment (Qiagen). The amount of RNA was quantified spectrophotometrically and 2 μg of total RNA from yeasts were reverse transcribed using the Superscript First-Strand synthesis system for RT-PCR (Invitrogen). The cDNA obtained was amplified by PCR using the specific primers described elsewhere (Martínez-Esparza, 2009). The amplicon was detected as a single band in an agarose gel in all the PCR reactions, the respective lengths of TPS1 and TPS2 being 70 and 60 bp. Real-time PCR was carried out using the SYBR-green method with β-actin as the internal standard. The Applied Biosystems 7500 Real–Time PCR System was used to run samples and analyse data. All samples were analysed in triplicate, normalized to the β-actin expression level and the results were expressed as fold induction compared with untreated controls.

Results

Sensitivity to external oxidants and trehalose storage in hog1 and cap1 mutants

In C. albicans, the stress-sensing MAP kinases Hog1 and Cap1 play an essential role in the defensive mechanisms against external oxidant aggressions (Alonso-Monge, 2003; Enjalbert, 2003, 2006; Smith, 2004). A hog1 null mutant was seen to be consistently very susceptible to increasing concentrations of both H2O2 and the superoxide-generating compound, menadione (Fig. 1). The homozygous cap1 mutant also showed high sensitivity, close to that of hog1, but slightly higher (Fig. 1). However, the most severe loss of cell viability corresponded to the double hog1cap1 null mutant (Fig. 1), confirming that this mutant is seriously compromised and therefore unable to overcome the oxidative stress caused by H2O2 or superoxide anions (Alonso-Monge, 2003; Smith, 2004). The reported phenotypes were counteracted by reintroducing the corresponding native HOG1 and CAP1 genes (data not shown).

1

Percentage of cell survival in Candida albicans hog1 and cap1 mutants after oxidative stress exposure, as well as during the adaptive oxidative response and during cross-tolerance. Cultures of the indicated strains were grown in YPD liquid medium at 37°C until they reached the exponential phase (OD600 nm=0.8–1.0). Identical samples were directly exposed for 60 min with gentle or acute doses of H2O2 (a) or menadione (MD) (b). Another sample was preincubated with 0.5 mM of either oxidant for 60 min and immediately challenged with the fungicidal concentration of the same compound, namely 50 mM H2O2 (a) or 40 mM MD (b), for the same time. Finally, a third sample was pretreated with 0.5 mM H2O2, followed by 40 mM MD (a) or with 0.5 mM MD, followed by 50 mM H2O2 (b). Aliquots were taken and appropriately diluted and spread on solid YPD in triplicate. Viability data were normalized to a control measurement (100%). The experiment was repeated twice in triplicate, with consistent results, and the values shown are the mean ± SD.

1

Percentage of cell survival in Candida albicans hog1 and cap1 mutants after oxidative stress exposure, as well as during the adaptive oxidative response and during cross-tolerance. Cultures of the indicated strains were grown in YPD liquid medium at 37°C until they reached the exponential phase (OD600 nm=0.8–1.0). Identical samples were directly exposed for 60 min with gentle or acute doses of H2O2 (a) or menadione (MD) (b). Another sample was preincubated with 0.5 mM of either oxidant for 60 min and immediately challenged with the fungicidal concentration of the same compound, namely 50 mM H2O2 (a) or 40 mM MD (b), for the same time. Finally, a third sample was pretreated with 0.5 mM H2O2, followed by 40 mM MD (a) or with 0.5 mM MD, followed by 50 mM H2O2 (b). Aliquots were taken and appropriately diluted and spread on solid YPD in triplicate. Viability data were normalized to a control measurement (100%). The experiment was repeated twice in triplicate, with consistent results, and the values shown are the mean ± SD.

We tested whether an adaptive response to oxidative stress is operative in hog1 and cap1 mutants; the existence of such a mechanism in C. albicans has been disputed (Jamieson, 1996; Alvarez-Peral, 2002; Enjalbert, 2003, 2006). Regardless of the specific disruption introduced, all the tested strains showed a notable recovery of cell viability when pertinent exponential cultures were preincubated at permissive doses (0.5 mM H2O2 or 0.5 mM menadione), followed by a challenge with severe concentrations of oxidants (50 mM H2O2 or 40 mM menadione) (Fig. 1a and b), albeit the percentage of survival was lower in the mutants than that in wild-type cells (Fig. 1a and b). This adaptive oxidative response seems to be a general feature shared by a set of different C. albicans genetic backgrounds (Jamieson, 1996; González-Párraga, 2003). Furthermore, when the hypothetical existence of oxidative cross-protection between the two oxidants was tested, it was found that cells treated with low doses (0.5 mM) of H2O2 or menadione acquired tolerance against subsequent acute exposure to either 40 mM menadione (Fig. 1a) or 50 mM H2O2 (Fig. 1b). Although varying in degree, this adaptive mechanism was manifested by all the tested strains (Fig. 1).

Unlike in budding and fission yeasts, the stress-protective accumulation of trehalose in C. albicans is specifically triggered by oxidative and heat-shock exposures, rather than by osmotic stress (Alvarez-Peral, 2002). Hence, we investigated the hypothetical connection between the oxidative responses mediated by Hog1 and Cap1 with the level of trehalose storage. The results presented in Table 1 indicate that a rapid and noticeable intracellular synthesis of trehalose occurs immediately after acute oxidative treatment in hog1- or in cap1-deficient cells, so that trehalose synthesis is not dependent on these signalling pathways or dependent on Hog1 or Cap1, because all the strains analysed showed an increase in their intracellular content of trehalose. Interestingly, hog1 null mutants always displayed a higher level of basal trehalose (in the absence of any stress) than the parental RM100 strain or the cap1 mutant (Table 1).

1

Effect of double disruption of hog1 and cap1 on the intracellular accumulation of trehalose in response to external oxidative stress (H2O2 and menadione), as well as during the adaptive response to oxidative stress in Candida albicans

Oxidative treatment Intracellular trehalose (nmolmg−1 wet wt) 
Strain 
RM100 hog1/hog1 cap1/cap1 hog1/hog1//cap1/cap1 
H2O2 Control 3.3 ± 0.2 5.8 ± 0.3 3.9 ± 0.2 5.5 ± 0.3 
0.5 mM 4.3 ± 0.2 7.3 ± 0.4 4.8 ± 0.3 6.7 ± 0.4 
50 mM 9.0 ± 0.5 16.3 ± 1.0 11.7 ± 0.7 15.5 ± 0.9 
0.5–50 mM 8.7 ± 0.5 14.9 ± 0.9 10.6 ± 0.6 12.2 ± 0.7 
Menadione 0.5 mM 2.5 ± 0.1 4.1 ± 0.2 2.8 ± 0.1 6.1 ± 0.3 
40 mM 17.7 ± 1.0 20.5 ± 1.2 18.9 ± 1.1 21.8 ± 1.2 
0.5–40 mM 12.9 ± 0.8 16.1 ± 0.7 11.9 ± 0.7 17.7 ± 1.0 
Oxidative treatment Intracellular trehalose (nmolmg−1 wet wt) 
Strain 
RM100 hog1/hog1 cap1/cap1 hog1/hog1//cap1/cap1 
H2O2 Control 3.3 ± 0.2 5.8 ± 0.3 3.9 ± 0.2 5.5 ± 0.3 
0.5 mM 4.3 ± 0.2 7.3 ± 0.4 4.8 ± 0.3 6.7 ± 0.4 
50 mM 9.0 ± 0.5 16.3 ± 1.0 11.7 ± 0.7 15.5 ± 0.9 
0.5–50 mM 8.7 ± 0.5 14.9 ± 0.9 10.6 ± 0.6 12.2 ± 0.7 
Menadione 0.5 mM 2.5 ± 0.1 4.1 ± 0.2 2.8 ± 0.1 6.1 ± 0.3 
40 mM 17.7 ± 1.0 20.5 ± 1.2 18.9 ± 1.1 21.8 ± 1.2 
0.5–40 mM 12.9 ± 0.8 16.1 ± 0.7 11.9 ± 0.7 17.7 ± 1.0 

Exponential yeast cells (OD600 nm=0.8–1.0) of the C. albicans strains used in this study: RM100, hog1/hog1, cap1/cap1 and hog1/hog1//cap1/cap1 were directly subjected to gentle (0.5 mM H2O2 and menadione) or severe (50 mM H2O2 or 40 mM menadione) oxidative exposure or preincubated for 60 min with low oxidative doses and immediately challenged with a fungicidal concentration (adaptive response). Samples for trehalose determination were harvested after 60 min of treatment. Results represent the mean ± SD of three independent determinations.

Heat shock-induced oxidative cross-protection is independent of Hog1

As mentioned above, the existence of a core stress response has also been convincingly proven in Saccharomyces cerevisiae and Schizosaccharomyces pombe. By means of this efficient ability, proliferating cells (preferentially) can resist potentially lethal injuries, when they are previously stimulated with mild doses of (in principle) nonrelated challenges. However, in C. albicans, the operability of this mechanism is still a disputed subject (Enjalbert, 2003, 2006), although we have found that in a trehalose-deficient mutant, an upshift from 28 to 37 °C improves the percentage of survival against further exposure with 50 mM H2O2 (Alvarez-Peral, 2002).

Here, the analysis is extended to the Hog1 and Cap1-mediated pathways. When actively growing cultures of the homozygous hog1 and cap1 mutants were treated for 60 min at 37 °C, they clearly showed an improvement in their capacity to withstand a subsequent challenge with 50 mM H2O2 compared with control samples kept at 28 °C (Fig. 2b). Notably, a significant degree of cell recovery was also conspicuous in the double hog1cap1 mutant, whose normal growth in the presence of a high dosage of oxidants is drastically impaired (Figs 1 and 2b). When the time-course activation pattern of the MAPKs Mkc1 and Hog1 in parental cells was followed upon thermal transfer from 28 to 37 °C, the temperature upshift was seen to cause changes in the MAPK phosphorylation state. Firstly, the Mkc1 MAP kinase, involved in the cell wall integrity pathway, is dephosphorylated and rephosphorylated after 40 min of incubation at 37 °C (Fig. 2a). Secondly, phosphorylated Hog1 was detected after 40 min of incubation (Fig. 2a). Accordingly, the temperature upshift triggered the reversible phosphorylation of both MAPKs after 1 h and before oxidative challenge. These data also support that C. albicans possesses a cross-tolerance response, which must be, at least in part, independent of Hog1 signalling.

2

An upshift in the incubation temperature leads to MAPK kinase phosphorylation (a) as well as to an increase in cell survival (cross-tolerance) to oxidative stress (b, c) in Candida albicans. (a) Cultures of the parental strain RM100 growing logarithmically at 28 or 37°C were transferred to 37 or 42°C, respectively. Samples were taken 0, 10, 40 and 60 min after the thermal transfer and processed for an immune-blot assay as indicated in Materials and methods. Hog1-P and Mkc1-P represent the phosphorylated form of these MAPKs. YPD-grown cultures of the strains under study were incubated at 28°C (b) or 37°C (c). When they reached the mid-log phase (OD600 nm=0.8), an aliquot was transferred from 28 to 37°C (b) or from 37 to 42°C (c) for 60 min. After returning to the initial temperature, an acute oxidative challenge (50 mM H2O2) was applied to both heat-shocked and control samples. For other details, see Fig. 1.

2

An upshift in the incubation temperature leads to MAPK kinase phosphorylation (a) as well as to an increase in cell survival (cross-tolerance) to oxidative stress (b, c) in Candida albicans. (a) Cultures of the parental strain RM100 growing logarithmically at 28 or 37°C were transferred to 37 or 42°C, respectively. Samples were taken 0, 10, 40 and 60 min after the thermal transfer and processed for an immune-blot assay as indicated in Materials and methods. Hog1-P and Mkc1-P represent the phosphorylated form of these MAPKs. YPD-grown cultures of the strains under study were incubated at 28°C (b) or 37°C (c). When they reached the mid-log phase (OD600 nm=0.8), an aliquot was transferred from 28 to 37°C (b) or from 37 to 42°C (c) for 60 min. After returning to the initial temperature, an acute oxidative challenge (50 mM H2O2) was applied to both heat-shocked and control samples. For other details, see Fig. 1.

Because C. albicans belongs to the usual microbiota found in humans (as a commensal), 37 °C being the standard temperature of the human body, considerable experimental work on Hog1 and Cap1 functions is usually conducted at 37 °C (Alonso-Monge, 1999; Navarro-García, 2005). Therefore, a transfer from 28 to 37 °C might not be considered, strictly speaking, a heat shock. As an alternative approach, the cross-protection response was also assayed by transferring exponentially growing YPD cultures from 37 to 42 °C (1 h) before applying a severe oxidative dosage (50 mM H2O2), this upshift being considered a true thermal stimulus. As shown in Fig. 2c, temperature increases within the range of growth compatibility led to a clear increase in the percentage of survival in the face of a high dosage of H2O2 in all the hog1 and cap1 null mutants checked. Except for the double hog1 cap1 mutant, the extent of cell recovery was similar in the parental and mutant strains (Fig. 2c); once again, the cross-protection phenotype of cap1 was roughly equivalent to that exhibited by hog1 (Fig. 2c).

Parallel measurements of endogenous trehalose revealed a higher basal content of the disaccharide upon transfer from 37 to 42 °C, while further application of 50 M H2O2 promoted a weak additional accumulation of trehalose (Table 2). We also confirmed the elevated basal content of trehalose resulting from HOG1 disruption in relation to the rest of the strains (Table 2). This heat shock caused a dephosphorylation of both Mkc1 and Hog1 MAPKs that was delayed compared with that observed for the 28 to 37 °C upshift. After 1 h of incubation, both MAPKs again became phosphorylated (Fig. 2a) (Navarro-García, 2005). Hence, during the application of treatments that lead to adaptive responses, a coordinated deactivation and reactivation of these two stress-responsive MAPKS seems to take place.

2

Changes in the endogenous trehalose content measured in the homozygous hog1 and cap1 mutants of Candida albicans during adaptive ‘cross-protection’ to a severe oxidative stress (50 mM H2O2) promoted by two different temperature upshifts (28→37 and 37→−42°C)

Growth Treatment Trehalose (nmol mg−1 wet wt) 
Strain 
RM100 hog1/hog1 cap1/cap1 hog1/hog1//cap1/cap1 
28°C Control 3.2 ± 0.2 4.1 ± 0.3 3.8 ± 0.3 3.9 ± 0.2 
H2O2 6.8 ± 0.5 11.1 ± 0.8 8.9 ± 0.6 9.7 ± 0.6 
37°C 5.8 ± 0.4 8.9 ± 0.6 5.1 ± 0.4 7.0 ± 0.5 
37°CH2O2 10.9 ± 0.8 15.5 ± 0.1 14.8 ± 1.0 15.8 ± 1.1 
37°C Control 4.2 ± 0.3 7.0 ± 0.5 4.6 ± 0.3 4.8 ± 0.3 
H2O2 8.9 ± 0.6 18.6 ± 1.3 10.0 ± 0.7 15.7 ± 1.0 
42°C 12.6 ± 0.9 20.7 ± 1.5 14.8 ± 1.0 19.9 ± 1.4 
42°CH2O2 15.2 ± 1.1 23.0 ± 1.6 16.4 ± 1.2 21.2 ± 1.5 
Growth Treatment Trehalose (nmol mg−1 wet wt) 
Strain 
RM100 hog1/hog1 cap1/cap1 hog1/hog1//cap1/cap1 
28°C Control 3.2 ± 0.2 4.1 ± 0.3 3.8 ± 0.3 3.9 ± 0.2 
H2O2 6.8 ± 0.5 11.1 ± 0.8 8.9 ± 0.6 9.7 ± 0.6 
37°C 5.8 ± 0.4 8.9 ± 0.6 5.1 ± 0.4 7.0 ± 0.5 
37°CH2O2 10.9 ± 0.8 15.5 ± 0.1 14.8 ± 1.0 15.8 ± 1.1 
37°C Control 4.2 ± 0.3 7.0 ± 0.5 4.6 ± 0.3 4.8 ± 0.3 
H2O2 8.9 ± 0.6 18.6 ± 1.3 10.0 ± 0.7 15.7 ± 1.0 
42°C 12.6 ± 0.9 20.7 ± 1.5 14.8 ± 1.0 19.9 ± 1.4 
42°CH2O2 15.2 ± 1.1 23.0 ± 1.6 16.4 ± 1.2 21.2 ± 1.5 

Two overnight cultures of the four strains grown at 28 or 37°C were refreshed in YPD to an OD of 0.2 and incubated at the same temperatures until they reached an OD600 nm=0.8–1.0. For each experiment, a sample was subjected to heat shock (28→37 or 37→42°C) for 60 min and immediately challenged with 50 mM H2O2. For other details, see Table 1.

H2O2 enhances the transcription of genes coding for trehalose biosynthesis

We analysed whether the striking synthesis of trehalose in the presence of oxidative stress was caused by a Hog1-dependent enhanced transcription of genes involved in trehalose biosynthesis (TPS1 and TPS2) or the consequence of a post-translational activation of the preformed biosynthetic enzymes. The time-course degree of gene expression induced by the supply of 5 mM H2O2 and quantified by real-time RT-PCR is shown in Fig. 3 (the application of 50 mM H2O2 was dismissed because of the severe cell toxicity). The results showed a rapid and noticeable mRNA increase in trehalose biosynthetic genes, which was independent of Hog1 and Cap1 and that followed quite similar kinetics of transcription in all the tested strains (Fig. 3). Both TPS1 and TPS2 mRNA expression was roughly similar, reaching a maximum 15 min after the addition of 5 mM H2O2, and reverted to control (nontreated) levels within 60 min (Fig. 3). These results are consistent with a direct transcriptional effect of H2O2 on the genes involved in trehalose biosynthesis, as well as with the suggestion that Tps1p and Tps2p proteins may be integrated into a single enzymatic complex, whose expression would be coordinated (Martínez-Esparza, 2009).

3

H2O2-induced mRNA expression of the trehalose biosynthetic genes is independent of Hog1 and Cap1. Exponential YPD-grown cells of the referred strains at 37°C were treated with 5 mM H2O2 for the indicated times. After extraction of total RNA, 2 μg was reverse transcribed and real-time q-PCR was performed using specific primers for TPS1 (upper panel) and TPS2 (lower panel). A value of 1 was assigned to the level recorded in untreated cells. For other details, see Materials and methods. Similar results were obtained in an independent experiment.

3

H2O2-induced mRNA expression of the trehalose biosynthetic genes is independent of Hog1 and Cap1. Exponential YPD-grown cells of the referred strains at 37°C were treated with 5 mM H2O2 for the indicated times. After extraction of total RNA, 2 μg was reverse transcribed and real-time q-PCR was performed using specific primers for TPS1 (upper panel) and TPS2 (lower panel). A value of 1 was assigned to the level recorded in untreated cells. For other details, see Materials and methods. Similar results were obtained in an independent experiment.

Antioxidant enzymatic activities in hog1 and cap1 mutants subjected to oxidative stress (H2O2)

The enhanced expression of antioxidant genes coding for enzymes possessing both protective and repairing functions represents a sharp response to the addition of external oxidants as well as to an early phase of genetic reprogramming after C. albicans–macrophages interaction (Lorenz, 2004; Enjalbert, 2006). Here, we provide additional biochemical support for this proposal by monitoring the activities of several specific antioxidant enzymes upon oxidative challenge with exogenous H2O2. Exponential cultures from all the strains under study subjected to gentle concentrations of H2O2 (0.5 and 5 mM) underwent a clear activation of catalase and GR (Fig. 4a and b). In turn, when severe oxidative conditions were introduced with 50 mM H2O2, a significant decay of both activities was recorded, presumably due to the severe cell damage caused by this toxic H2O2 treatment (Fig. 4). Curiously, although these two antioxidant enzymes exhibited lower basal activities in the hog1 and hog1cap1 mutants with respect to the wild type, the magnitude of enzymatic activation triggered by oxidative stress was slightly greater in hog1 and cap1 cells, providing additional support for the existence in C. albicans of a compensatory mechanism independent of Hog1 and Cap1, but that is insufficient to ensure the maintenance of cell viability (Alvarez-Peral, 2002; Alonso-Monge, 2009).

4

Effect of external addition of different doses of H2O2 for 60 min on the levels of catalase (a) and GR (b) activities in Candida albicans. Mid-log cultures of the parental strain RM100, hog1 and cap1 mutants were taken and processed as described in Materials and methods. Activity data are expressed with respect to an untreated control. Values represent the means ± SD of three determinations.

4

Effect of external addition of different doses of H2O2 for 60 min on the levels of catalase (a) and GR (b) activities in Candida albicans. Mid-log cultures of the parental strain RM100, hog1 and cap1 mutants were taken and processed as described in Materials and methods. Activity data are expressed with respect to an untreated control. Values represent the means ± SD of three determinations.

The antioxidant behaviour of SOD was also verified in HOG1- and CAP1-deleted strains. As a consequence of its intrinsic dismutative action on the toxic O2, the SOD isozymes are involved in the virulence of C. albicans and protection against endogenous ROS (Hwang, 2002). An increase in global SOD activity was recorded in the hog1 and hog1cap1 mutants after the oxidative treatments, whose extent varied with the intensity of the stress (Fig. 5a), whereas in RM100 and cap1 cells, SOD was only activated upon weak exposure to 0.5 mM H2O2 (Fig. 5a). However, the pattern of SOD isozyme activity changed as a function of the doses applied. At mild nonlethal concentrations (0.5–5 mM H2O2), the activation corresponded to cytosolic Cu,Zn-SOD (Fig. 5b), which would presumably detoxify the H2O2 that permeates through the plasma membrane; however, in severe oxidant treatments, the mitochondrial Mn-SOD was the predominant isoform contributing to the whole activity (Fig. 5c), being especially evident in the strains lacking the HOG1 gene. Interestingly, this mitochondrial Mn-SOD isozyme represents the first line of defence against superoxide radical anions formed as products of oxidative phosphorylation. Unlike cytosolic Cu,Zn-SOD, the mitochondrial SOD is not involved in virulence (Hwang, 2002).

5

Changes in the total SOD activity (a), Cu,Zn isozyme (b) and Mn-SOD isozyme (c) in Candida albicans Hog1- and Cap1-deficient mutants subjected to oxidative stress with low and high H2O2 concentrations. The three enzymatic activities were determined simultaneously in the same sample following the reported protocols. For other details, see Fig. 4.

5

Changes in the total SOD activity (a), Cu,Zn isozyme (b) and Mn-SOD isozyme (c) in Candida albicans Hog1- and Cap1-deficient mutants subjected to oxidative stress with low and high H2O2 concentrations. The three enzymatic activities were determined simultaneously in the same sample following the reported protocols. For other details, see Fig. 4.

Discussion

The defensive mechanisms against oxidative stress are undoubtedly of major relevance in microbial pathogens, because the generation of potentially lethal levels of ROS by phagocytic cells has been demonstrated to be a major challenge encountered during the first steps of an infection (Vazquez-Torres & Balish, 1997; Fang, 2004). Candida albicans is the most prevalent fungal pathogen in humans and, after being phagocytosed, it has to encounter the high levels of ROS released inside macrophages and neutrophils (Vazquez-Torres & Balish, 1997; Fang, 2004; Chauhan, 2006). Despite the strong evidence supporting the existence of a core stress response in C. albicans (Smith, 2004; Enjalbert, 2006), emerging data point to specific and finely regulated oxidative-responsive mechanisms, not always shared by other environmental stress (Enjalbert, 2003, 2007; Lorenz, 2004; Alonso-Monge, 2009). In fact, a transcriptional analysis demonstrates an increased expression of the genetic machinery necessary for both DNA damage repair and antioxidant protection during the early genetic reprogramming that takes place after C. albicans ingestion by macrophages (Lorenz, 2004).

The Hog1 signalling pathway plays a pivotal role in the control of defensive responses to oxidative stress in C. albicans, and the double disruption of the HOG1 gene causes hypersensitivity to external oxidants (Alonso-Monge, 1999, 2003; Smith, 2004) (Fig. 1); this outcome may be relevant in virulence. In fact, hog1 null cells show a high degree of susceptibility to attack by phagocytic cells, the PMNs having a predominant antimicrobial function as regards the macrophages (Arana, 2007; Enjalbert, 2007). However, in the case of C. albicans, activation of a specific set of antioxidant gene reporters is a niche-specific event, because the threat of oxidative killing is no longer significant once a productive infection has been established, at least in the kidney (Enjalbert, 2007). Remarkably, Hog1 also appears to be involved in the respiratory metabolism and hog1 null mutants also display enhanced basal mitochondrial activity and produce elevated intracellular levels of ROS, despite an increased catalase activity (Alonso-Monge, 2003, 2009). Apart from the Hog1-mediated responses, in the cell protection against oxidative stress also contributes the bZIP transcription factor Cap1 (the homologue of S. cerevisiae Yap1) (Alarco & Raymond, 1999). Hog1 and Cap1 seem to act through different, but otherwise complementary, mechanisms (Alonso-Monge, 2003; Smith, 2004).

In contrast, this study provides consistent data on the existence in C. albicans of Hog1- and Cap1-independent defensive mechanisms in the face of oxidative aggression. Thus, wild-type growing cultures exposed to toxic levels of H2O2 stored large amounts of intracellular trehalose, an effect not altered by the disruption of either Hog1 or Cap1 (Table 1), even though this rapid response could not counteract the severe cell killing recorded in the two homozygous mutants (Fig. 1). Accordingly, mRNA expression in the trehalose biosynthetic genes (TPS1 and TPS2) followed fairly identical kinetics in both parental and mutant cultures (Fig. 3). Interestingly, hog1 cells seem to compensate their drastic metabolic defects by increasing the basal levels of other protectants, such as trehalose (Tables 1 and 2). This result is also consistent with the elevated activity of thioredoxin reductase (TRR1) shown by hog1 cells in the absence of oxidants (Arana, 2007). The drastic loss of viability suffered by the double hog1 cap1 mutant favours the idea that the two pathways sense and respond to distinct stimuli through independent ways (Alonso-Monge, 2003; Smith, 2004).

Furthermore, neither the adaptive responses to oxidative stress (menadione and H2O2) nor the cross-protection (from temperature to oxidative stress) are mediated by Hog1 and Cap1 (Figs 1 and 2). Both processes may deserve great importance for the successful colonization of deep tissues and organs during the course of an infection. Because the respiratory burst is triggered upon the pathogens' engulfment by phagocytic cells, a gradual adaptation allows them to cope with the increasing concentrations of ROS inside the phagosomes, avoiding its destruction (Fang, 2004; Chauhan, 2006; Arana, 2007). In turn, the oxidative stress contributes to the lethal effect of heat shock in budding yeasts (Davidson, 1996). Regardless of any Hog1 and Cap1 genotype, preincubation with a low dosage of H2O2 and menadione confers a clear improvement in the level of cell recovery determined by counting the fraction of viable cells (Fig. 1). Although trehalose accumulation occurs during adaptive oxidative exposure (Table 1), the disaccharide does not seem to play a protective role during these adaptive responses induced (Alvarez-Peral, 2002).

Smith (2004) postulated that the acquisition of cross-protection in C. albicans between different stresses is under the control of Hog1 and only takes place if Hog1p is phosphorylated and translocated to the nucleus. In this sense, a temperature increase from 23 to 37 °C leads to Hog1p dephosphorylation, and so the subsequent induction of a cross-protective mechanism does not take place (Smith, 2004). In our case, increases in temperature led to changes in the phosphorylation pattern of the stress-responsive MAPKs, Mkc1 and Hog1 (Fig. 2a). This fact demonstrates that both MAPKs are relevant for sensing and adapting to external changes and that signalling transduction involves coordinated multifactorial mechanisms. Moreover, these thermal upshifts exerted a clear protective effect when the cultures were further faced with an acute oxidative stress. This cross-tolerance allowed a significant recovery in cell survival (Fig. 2b). Hence, cross-protection involves mechanisms that are, at least in part, independent of Hog1 and Cap1.

The transcriptional induction of oxidative stress-responsive genes (Enjalbert, 2006) appears to correlate with measurements of antioxidant enzymatic activities (Figs 4 and 5). The extent of enzymatic activation was always more pronounced in hog1 and cap1 mutant cells than in wild-type cells (Figs 4 and 5), as occurs in a trehalose-deficient mutant (González-Párraga, 2003). The severity of the oxidative challenge imposed determines the nature of the measured response. Thus, moderate H2O2 exposures (0.5 and 5 mM) promoted a marked activation of catalase and GR activities, whereas a drastic treatment (50 mM H2O2) was deleterious for catalase, but did not affect the control levels of GR activity (Fig. 4). In turn, SOD exhibited a differential pattern of isozyme activation, cytosolic Cu, Zn accounting for most of the SOD activity observed in low oxidative treatments, while this activity was replaced by mitochondrial Mn-SOD at potentially toxic doses of H2O2 (Fig. 5). This enzyme is responsible for detoxifying the O2 generated through oxidative phosphorylation (Hwang, 2002), and its elevated activity upon acute oxidative stress (Fig. 5) correlates with the enhanced production of ROS observed in the hog1 mutant (Alonso-Monge, 2003). Taken together, our results are consistent with the existence in C. albicans of a complex defensive machinery against oxidative stress, in which both Hog1- and Cap1-dependent and -independent mechanisms are involved.

Authors' contribution

P.G.-P. and R.A.-M. contributed equally to this paper.

Acknowledgements

The experimental work was supported by grants BIO-BMC 06/01-0003 from Dirección General de Investigación (Comunidad de Murcia, Spain), BFU2006-08684/BMC (Ministerio de Educación y Ciencia, Spain), BIO2006-03637 (Programa Nacional de Biotecnología) and S-SAL/0246/2006 from Comunidad de Madrid (Spain). We are also indebted to the financial contract provided by Cespa-Ingeniería Urbana, S.A. (Spain).

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Author notes

Editor: José Ruiz-Herrera