Fluconazole and amphotericin-B resistance are associated with increased catalase and superoxide dismutase activity in Candida albicans and Candida dubliniensis

Abstract

Introduction

Candida dubliniensis, a new species of Candida that has been recovered from several sites in healthy people, has been associated with recurrent episodes of oral candidiasis in AIDS and HIV-positive patients. This species is closely related to C. albicans. The enzymatic activity of C. dubliniensis in response to oxidative stress is of interest for the development of drugs to combat C. dubliniensis.

Methods

Fluconazole- and amphotericin B-resistant strains were generated as described by Fekete-Forgács et al. (2000). Superoxide dismutase (SOD) and catalase assays were performed as described by McCord and Fridovich (1969) and Aebi (1984), respectively.

Results

We demonstrated that superoxide dismutase (SOD) and catalase activities were significantly higher (p<0.05) in the fluconazole- and amphotericin B-resistant strains of C. dubliniensis and C. albicans than in the sensitive strains. The catalase and SOD activities were also significantly (p<0.01) higher in the sensitive and resistant C. albicans strains than in the respective C. dubliniensis strains.

Conclusions

These data suggest that C. albicans is better protected from oxidative stress than C. dubliniensis and that fluconazole, like amphotericin B, can induce oxidative stress in Candida; oxidative stress induces an adaptive response that results in a coordinated increase in catalase and SOD activities. [PUBLICATION ABSTRACT]

Major Articles

Fluconazole and amphotericin-B resistance are associated with increased catalase and superoxide dismutase activity in Candida albicans and Candida dubliniensis

Carlos Eduardo Blanco Linares[1], Sandro Rogério Giacomelli[2], Delsi Altenhofen[2], Sydney Hartz Alves[3], Vera Maria Morsch[4], Maria Rosa Chitolina Schetinger[4]

[1]Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS

[2]Curso de Farmácia, Centro de Ciências da Saúde, Universidade Regional do Alto Uruguai e das Missões, Frederico Westphalen, RS

[3]Departamento de Microbiologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, RS

[4]Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS

ABSTRACT

Introduction

Candida dubliniensis, a new species of Candida that has been recovered from several sites in healthy people, has been associated with recurrent episodes of oral candidiasis in AIDS and HIV-positive patients. This species is closely related to C. albicans. The enzymatic activity of C. dubliniensis in response to oxidative stress is of interest for the development of drugs to combat C. dubliniensis.

Methods

Fluconazole- and amphotericin B-resistant strains were generated as described by Fekete-Forgács et al. (2000). Superoxide dismutase (SOD) and catalase assays were performed as described by McCord and Fridovich (1969) and Aebi (1984), respectively.

Results

We demonstrated that superoxide dismutase (SOD) and catalase activities were significantly higher (p<0.05) in the fluconazole- and amphotericin B-resistant strains of C. dubliniensis and C. albicans than in the sensitive strains. The catalase and SOD activities were also significantly (p<0.01) higher in the sensitive and resistant C. albicans strains than in the respective C. dubliniensis strains.

Conclusions

These data suggest that C. albicans is better protected from oxidative stress than C. dubliniensis and that fluconazole, like amphotericin B, can induce oxidative stress in Candida; oxidative stress induces an adaptive response that results in a coordinated increase in catalase and SOD activities.

Keywords: Catalase; Superoxide dismutase; Candida albicans ; Candida dubliniensis

INTRODUCTION

Candida dubliniensis, a new emerging opportunistic species, was first described in 1995 in Dublin, Ireland. The cultures were obtained from human immunodeficiency virus (HIV)-positive patients with recurrent oral candidiasis. Although C. dubliniensis was initially recovered from the oral cavity, this species has also been reported at other body sites, including the respiratory tract, central nervous system, vagina, and skin, as well as in the blood, urine, and feces of both HIV-positive and HIV-negative patients1-4. Nunn et al.5 also described the isolation of C. dubliniensis from nonhuman environmental sources. Thus, this species is not confined to humans5.

Candida dubliniensis is phenotypically and genotypically similar to C. albicans and C. stellatoidea. Like these Candida species, C. dubliniensis forms chlamydospores and is germ tube-positive in phenotypic identification tests. The detection of unusual carbohydrate assimilation profiles, which can be identified using commercially available systems such as the API® ID 32C and API® 20C AUX kits, were one of the earliest indications that C. dubliniensis represented a new species within the Candida genus4. Studies have since documented a high prevalence of this organism in patients suffering from diabetes (3.6 to 18.2%), cystic fibrosis (11.1%), cancer (2 to 4.6%), vulvovaginal candidiasis (0.17 to 2.4%), and candidemia (0.5 to 7.9%)6.

Candida dubliniensis is more susceptible to polyenes and azoles than C. albicans; however, a remarkable feature of C. dubliniensis is the rapid development of secondary resistance to fluconazole7. Azoles, such as fluconazole, act on the ergosterol biosynthesis pathway by inhibiting the enzyme C14-?-demethylase, a cytochrome P450-dependent enzyme8. Amphotericin B, a polyene antifungal agent, binds to sterols such as ergosterol and disrupts the osmotic integrity of the fungal membrane, resulting in the leakage of intracellular ions and metabolites9. Despite intense study of C. dubliniensis, the enzymatic activities of superoxide dismutase (SOD) and catalase in fluconazole-resistant and amphotericin B-resistant C. dubliniensis are unknown. In Candida, SOD plays an important protective role against attack by macrophages and neutrophils, which generate superoxide radicals10. Superoxide dismutase converts damaging superoxide radicals (O2·-), one type of reactive oxygen species (ROS), to the less damaging hydrogen peroxide (H2O2), which can be converted into water by catalase11,12. Catalase has been implicated in amphotericin B resistance by inhibiting the oxidative damage produced by this polyene13. Ergosterol, a sterol present in the fungal plasma membrane, is the target of amphotericin B. The binding of amphotericin B to ergosterol produces channels that leak potassium ions, resulting in the disruption of the proton gradient14,15. The antioxidant enzymes SOD and catalase may act synergistically to protect cells from oxidative damage16. Some authors have suggested that the coordinated inactivation of ROS by the sequential action of superoxide- and hydrogen peroxide-inactivating enzymes is essential for the effective protection of cells against ROS toxicity17.

To further explore the antioxidant enzymatic activities and the coordinated inactivation of ROS by SOD and catalase in C. dubliniensis and C. albicans, we investigated the SOD and catalase activities in fluconazole- and amphotericin B-sensitive and resistant strains of C. dubliniensis and C. albicans.

METHODS

Strains

The Candida albicans and Candida dubliniensis strains used in this study [Ca 10, Ca 81, Ca 119, Ca 170, Ca ATCC 44373, Cd 1, Cd 2, Cd 3, Cd 4, Cd 5, Cd 7, Cd 9, Cd 11, and Cd 13] were isolated from patients with oral candidiasis at the University Hospital of Universidade Federal de Santa Maria (Brazil). Species identification was based on phenotypic and genotypic characteristics. The C. dubliniensis genotype was confirmed by random amplification of polymorphic deoxyribonucleic acid (DNA) using the primers CDU (5? GCG ATC CCC A 3?) and B-14 (5? GAT CAA GTC C 3?)18,4. The C. albicans strain ATCC 44373 from the American Type Culture Collection was used as a control strain.

Generation of fluconazole-resistant strains

The minimal inhibitory concentration (MIC) of fluconazole was first determined for C. albicans and C. dubliniensis according to the Clinical and Laboratory Standards Institute (CLSI) document M27-A319. After the MIC determinations, the cultures were grown overnight in Sabouraud glucose broth (SDB), and the cells were added to flasks containing 20ml of media to achieve a final absorbance of 0.1 at 640nm. The cultures were incubated at 30°C for 10h, and fluconazole was then added to a final concentration of 8µg ml-1. After 14h of further incubation, the cells of the fluconazole-containing culture were consecutively subcultured 3 times in fresh SDB medium containing 8µg ml-1 fluconazole; in each case, the cultures were incubated at 30°C with shaking for 24h. After the third incubation, the cells were added to flasks containing 20ml of SDB medium containing 8µg ml-1 fluconazole, and the turbidity of the cultures was adjusted to achieve a final absorbance of 0.1 at 640nm. After a 10h incubation, fluconazole was added at a final concentration of 16µg ml-1, and after 14h, the cells from this culture were subcultured 3 times in fresh SDB medium containing 16µg ml-1 fluconazole; each subculture was incubated at 30°C with shaking for 24h. The fluconazole concentration was always duplicated under this procedure, and the final concentration was 64µg ml-1. Cells from this culture were plated on Sabouraud glucose agar (SDA), and the colonies were designated C. albicans fluconazole resistant and C. dubliniensis fluconazole resistant20.

Generation of amphotericin B-resistant strains

Induction of amphotericin B resistance was performed as described for the generation of the fluconazole-resistant strains, with some modifications. The time needed for cell growth between exposures to different amphotericin B concentrations was 48h. Resistant cells obtained at 2µg ml-1 were plated on SDA, and the colonies were designated C. albicans amphotericin B-resistant and C. dubliniensis amphotericin B-resistant.

Susceptibility tests

Antifungal agents: standard fluconazole (Pfizer Central Research) and amphotericin B (Bristol-Myers, Squibb Australia Pty Ltd) powders were obtained from their respective manufacturers. Stock solutions were prepared in water (fluconazole) and dimethyl sulfoxide (amphotericin B). Serial two-fold dilutions were prepared exactly as outlined in the CLSI document M27-A319. Final dilutions were made in Roswell Park Memorial Institute (RPMI) 1640 medium buffered to pH 7.0 with 0.165mmol l-1 morpholinopropanesulfonic acid (MOPS). Aliquots (100µl) of each antifungal agent at a 2X final concentration were dispensed into the wells of plastic microdilution trays, which were sealed and frozen at -70°C until use. The fluconazole dilutions ranged from 0.12 to 128µg ml-1, and the amphotericin B dilutions ranged from 0.0313 to 16µg ml-1.

Inoculum standardization - inoculum suspensions of the yeast strains were prepared in sterile saline (0.85% NaCl) from 24h cultures grown on SDA at 35°C. The turbidity of the suspensions was adjusted to achieve a final absorbance of 0.1 at 530nm8,19.

CLSI broth microdilution method (document M27-A3): microplates containing 100µl of two-fold serial dilutions of the antifungal drugs in standard RPMI 1640 medium (0.2% glucose) were inoculated with 100µl of inoculum containing 1 × 103 to 5 × 103 colony-forming units per milliliter (CFU/ml). Following inoculation, the reference microdilution plates were incubated at 35°C, and the Minimum Inhibitory Concentration MICs were determined after 24 and 48h. The fluconazole and amphotericin B reference MICs corresponded to the lowest drug dilutions that resulted in prominent growth inhibition (? 80%) or the absence of growth, respectively19.

Enzyme assays

Candida albicans and Candida dubliniensis strains: strains were cultured for 48h in SDA containing 64µg ml-1 fluconazole or 2µg ml-1 amphotericin B. The yeast cells were added to sterile saline (0.85%) to achieve a final absorbance of 0.1 at a wavelength of 625nm. Aliquots (1ml) of this suspension were added to 50ml of SDB containing 4% glucose and 1% mycological peptone, and these cultures were incubated at 30°C with shaking for 24h for the fluconazole experiments and for 48h for the amphotericin B experiments. After centrifugation, the cells were washed 3 times with sterile water, and a crude extract was prepared.

Preparation of cell-free extracts: the crude extracts were prepared using the glass bead lysis method. Cells that had been cultured in SDB supplemented with fluconazole or amphotericin B were washed 3 times with MilliQ water and resuspended in lysis buffer (50mmol l-1 potassium phosphate pH 7.0) containing 0.5g of 500-µm-diameter glass beads. The mixture was homogenized in a Teflon-glass homogenizer for 6 cycles of alternating homogenization for 20s and cooling on ice. The mixture was then centrifuged for 30 min in a refrigerated centrifuge to remove the cell debris and glass beads. The supernatant was used for enzyme assays.

Superoxide dismutase activity

Superoxide dismutase activity was assayed according to McCord and Fridovich21. The assay was performed in a total volume of 1ml containing 50mmol l-1 glycine buffer (pH 10), 60mmol l-1 epinephrine, and the enzyme. Epinephrine was added, and adrenochrome formation was recorded at 480nm with a ultraviolet-visible (UV-Vis) spectrophotometer for 4 min. One unit of SOD activity was equivalent to the amount of enzyme required to inhibit epinephrine oxidation by 50% under the experimental conditions. The assays were performed in triplicate.

Catalase activity

Catalase activity was determined in the cell-free extracts using the method of Aebi et al.22. H2O2 solution (10mM), enzyme extract, and 50mM phosphate buffer (pH 7) were pipetted into a cuvette. The reduction of H2O2 was followed at a wavelength of 240nm for 4 min against a blank containing 50mM phosphate buffer and enzyme extract. Catalase activity was expressed in ?E min-1 (mg protein)-1. The assays were performed in triplicate.

Protein analysis

Protein concentrations were measured using the Bradford method and Coomassie Blue; serum albumin was used as the standard23.

Statistics

Paired t tests were used to compare the sensitive and resistant cells. Comparisons among the C. albicans and C. dubliniensis groups were made using unpaired t tests. The calculations were performed using the GraphPad InStat statistical program.

Ethical considerations

This protocol was approved by the Bioethics Committee of the Universidade Regional Integrada do Alto Uruguai e das Missões from Frederico Westphalen - RS, Brazil, under registration n° 061-2/PIH/04.

RESULTS

Fluconazole- and amphotericin B-resistant C. dubliniensis and C. albicans strains were obtained by exposing sensitive isolates to increasing concentrations of these antifungal agents. For the sensitive C. dubliniensis cells, the MICs ranged from 0.06 to 0.5µg ml-1 for fluconazole and from 0.0312 to 0.125µg ml-1 for amphotericin B; for the sensitive C. albicans cells, the MICs ranged from 0.5 to 4.0µg ml-1 for fluconazole and from 0.0625 to 0.25µg ml-1 for amphotericin B. After the induction of resistance, tests of the susceptibility to fluconazole and amphotericin B demonstrated that resistance developed in all strains assayed.

Table 1 shows the SOD and catalase activities in the cell-free extracts of the fluconazole-sensitive and fluconazole-resistant C. dubliniensis strains. In general, the mean SOD activity was 1.97-fold greater for fluconazole-resistant C. dubliniensis than for the sensitive cells. The difference between the 2 groups was significant (p < 0.01). The catalase activity of the fluconazole-resistant cells was 1.58-fold greater than that of the fluconazole-sensitive cells; this difference was significant (p < 0.01).

TABLE 1 - Superoxide dismutase and catalase activities of the fluconazole-sensitive and fluconazole-resistant Candida dubliniensis strains.

Strains Mean SOD activity [U mg protein-1] Mean catalase activity [?E min-1 (mg protein)-1]

sensitivea resistantb,c sensitivea resistantb,d

01 13.4 ± 6.0 48.8 ± 2.8 0.5 ± 0.2 1.6 ± 0.1

02 56.4 ± 17.7 113.6 ± 23.6 1.9 ± 0.4 3.7 ± 0.1

03 27.1 ± 3.6 21.9 ± 5.2 0.8 ± 0.2 1.1 ± 0.1

04 5.1 ± 2.9 10.3 ± 2.5 0.5 ± 0.1 0.6 ± 0.1

05 17.2 ± 11.8 130.2 ± 33.7 1.5 ± 0.3 3.4 ± 0.5

07 14.0 ± 2.2 17.7 ± 3 1.1 ± 0.05 1.7 ± 0.4

09 28.2 ± 2.9 38.8 ± 12.2 0.6 ± 0.05 0.9 ± 0.2

11 7.4 ± 1.2 47.2 ± 6.1 1.9 ± 0.6 1.6 ± 0.7

13 83.2 ± 5.8 67.9 ± 6.2 1.5 ± 0.4 1.7 ± 0.7

The data are the mean ± SD of triplicate experiments. U: unit of SOD; ?E: absorbance change at 240nm over 240s.

a

Fluconazole-sensitive C. dubliniensis;

b

Fluconazole-resistant C. dubliniensis (64µg/ml-1);

c

Statistical analyses revealed significant differences between the SOD activities of the fluconazole-resistant and fluconazole-sensitive C. dubliniensis groups (p < 0.01);

d

Statistical analyses revealed significant differences between the catalase activities of the fluconazole-resistant and fluconazole-sensitive C. dubliniensis groups (p < 0.01).

SOD: superoxide dismutase; SD: standard deviation; C.: Candida.

Table 2 shows the SOD and catalase activities of the C. dubliniensis strains resistant to 2µg ml-1 amphotericin B. The mean SOD activity of the C. dubliniensis strain resistant to 2µg ml-1 amphotericin B was 1.97-fold greater than that of the sensitive cells; this difference was statistically significant (p < 0.01). The mean catalase activity of the group resistant to 2µg ml-1 amphotericin B was 2-fold greater than that of the sensitive group. Statistical analyses showed significant differences (p < 0.01) between the C. dubliniensis catalase activities of the cells resistant to 2µg ml-1 amphotericin B and the sensitive cells.

TABLE 2 - Superoxide dismutase and catalase activities of the amphotericin B-sensitive and amphotericin B-resistant Candida dubliniensis strains.

Strains Mean SOD activity [U mg protein-1] Mean catalase activity [?E min-1 (mg protein)-1]

sensitivea resistantb,c sensitivea resistantb,d

01 15.5 ± 0.4 69.7 ± 20.7 0.6 ± 0.1 2.4 ± 0.5

02 15 ± 1.5 22.6 ± 3.0 0.8 ± 0.1 0.9 ± 0.1

03 13.5. ± 0.4 37.8 ± 6.8 0.9 ± 0.01 2.1 ± 0.1

04 27.2 ± 1.8 50.7 ± 5.0 1.1 ± 0.1 1.8 ± 0.01

05 22.1 ± 3.2 37.3 ± 3.5 1.0 ± 0.1 1.8 ± 0.2

07 15.7 ± 1.9 44.1 ± 13.3 1.1 ± 0.01 3.1 ± 0.01

09 47.2 ± 12.6 101.1 ± 24.1 1.6 ± 0.2 2.2 ± 0.3

11 57.9 ± 33.2 69.3 ± 4.7 1.7 ± 0.2 3.6 ± 0.6

13 35.5 ± 10.2 57.8 ± 8.1 0.8 ± 0.1 1.0 ± 0.1

The data are the mean ± SD of triplicate experiments; U: unit of SOD; ?E: absorbance change at 240nm over 240s.

a

Amphotericin B-sensitive C. dubliniensis;

b

C. dubliniensis resistant to 2µg/ml-1 amphotericin B;

c

Statistical analyses indicated significant differences between the SOD activities of the C. dubliniensis cells resistant to 2µg/ml-1 amphotericin B and the sensitive C. dubliniensis cells (p < 0.01).

d

Statistical analyses indicated significant differences between the catalase activities of the C. dubliniensis cells resistant to 2µg/ml-1 amphotericin B and the sensitive C. dubliniensis cells (p < 0.01).

SOD: superoxide dismutase; SD: standard deviation; C.: Candida.

The SOD and catalase activities of the fluconazole-sensitive and fluconazole-resistant C. albicans cells are shown in Table 3. The mean SOD activity of the fluconazole-resistant C. albicans was 1.3-fold greater than that of the sensitive cells; this difference was statistically significant (p < 0.01). The mean catalase activity of the fluconazole-resistant group was 1.57-fold greater than that of the fluconazole-sensitive group; this difference was statistically significant (p < 0.01).

TABLE 3 - Superoxide dismutase and catalase activities of the fluconazole-sensitive and fluconazole-resistant Candida albicans strains.

Strains Mean SOD activity [U mg protein-1] Mean catalase activity [?E min-1 (mg protein)-1]

sensitivea resistantb,c sensitivea resistantb,d

Ca 10 177.4 ± 22.1 300.5 ± 2 2.8 ± 0.2 3.5 ± 0.6

Ca 81 230.4 ± 5.1 273.1 ± 2.8 2.2 ± 0.1 2.3 ± 0.4

Ca 119 269.6 ± 5.9 289.7 ± 21.3 1.5 ± 0.2 2.6 ± 0.3

Ca 170 139.2 ± 6.2 238.3 ± 16.9 1.6 ± 0.4 4.1 ± 0.2

Ca ATCC 44373 188 ± 2.5 205.3 ± 7 2.3 ± 0.2 3.8 ± 0.9

The data are the mean ± SD of triplicate experiments. U: unit of SOD; ?E: absorbance change at 240nm over 240s.

a

Fluconazole-sensitive C. albicans;

b

Fluconazole-resistant C. albicans (64µg/ml-1);

c

Statistical analyses revealed significant differences between the SOD activities of the fluconazole-resistant and fluconazole-sensitive C. albicans groups (p < 0.01);

d

Statistical analyses revealed significant differences between the catalase activities of the fluconazole-resistant and fluconazole-sensitive C. albicans groups (p < 0.01).

SOD: superoxide dismutase; SD: standard deviation; C.: Candida.

Table 4 shows the SOD and catalase activities of the C. albicans strains that were sensitive or resistant to 2µg ml-1 amphotericin B. The mean SOD activity of the C. albicans strain resistant to 2µg ml-1 amphotericin B was 1.97-fold greater than that of the sensitive group; this difference was statistically significant (p < 0.01). The mean catalase activity of the cells resistant to 2µg ml-1 amphotericin B was 1.77-fold greater than that of the sensitive group; this difference was statistically significant (p < 0.05).

TABLE 4 - Superoxide dismutase and catalase activities of the amphotericin B-sensitive and resistant Candida albicans strains.

Strains Mean SOD activity [U mg protein-1] Mean catalase activity [?E min-1 (mg protein)-1]

sensitivea resistantb,c sensitivea resistantb,d

Ca 10 22.1 ± 8.5 178.4 ± 15 3.1 ± 0.3 4.4 ± 0.3

Ca 81 116.2 ± 6.4 93 ± 4.8 2 ± 0.2 2.9 ± 0.3

Ca 119 106.6 ± 11.7 298.2 ± 21.3 1.5 ± 0.2 2.9 ± 0.8

Ca 170 186.1 ± 3.2 195 ± 6.9 1.3 ± 0.4 3.9 ± 0.1

Ca ATCC 44373 82.4 ± 4 269.3 ± 10.1 2.8 ± 0.1 4.8 ± 1.1

The data are the mean ± SD of triplicate experiments. U: unit of SOD. ?E: absorbance change at 240nm over 240s.

a

Amphotericin B-sensitive C. albicans;

b

C. albicans resistant to 2 µg ml-1 amphotericin B;

c

Statistical analyses revealed significant differences between the SOD activities of the C. albicans cells resistant to 2µg/ml-1 amphotericin B and the sensitive C. albicans cells (p < 0.01);

d

Statistical analyses revealed differences between the catalase activities of the C. albicans cells resistant to 2µg/ml-1 amphotericin B and the sensitive C. albicans cells (p < 0.05).

SOD: superoxide dismutase; SD: standard deviation; C.: Candida.

DISCUSSION

Since the recognition of C. dubliniensis as a new species in 1995, many studies have investigated the differences between this species and C. albicans. Studies of the responses of these two species under identical conditions have uncovered phenotypic as well as genotypic differences between these two species. Enzymes involved in virulence, such as aspartyl proteinases, phospholipases, chondroitin sulfatase, and hyaluronidase, have been thoroughly studied. De Bernardis et al.24 and Linares et al.25 demonstrated that the level of aspartyl proteinase was significantly higher in C. albicans than in C. dubliniensis, while no differences in phospholipase, chondroitin sulfatase, and hyaluronidase activities were observed24-26. In this study, we investigated the effect of fluconazole and amphotericin B resistance on the SOD and catalase activities of C. albicans and C. dubliniensis. These enzymes are not related to virulence factors but play important roles in the oxidative stress response in both species27. SOD activity is involved in the detoxification of free superoxide radicals, such as the radical produced by phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase29. Phagocytic cells have 2 antimicrobial systems that are responsible for the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). To counter these antimicrobial systems, microorganisms such as Candida have multiple defense responses that can be adjusted according to the nature and amount of ROS produced by the host or environment30. Our results revealed that SOD activity was significantly increased in C. dubliniensis and C. albicans strains that were resistant to amphotericin B and fluconazole compared to sensitive strains. The increase in SOD activity in response to amphotericin B may be due to the production of oxidative damage in Candida through lipid peroxidation15. In our study, yeast cultures were exposed to increasing concentrations of amphotericin B, up to a maximum of 2 µg ml-1. In general, at this concentration, amphotericin B kills Candida by inducing oxidative damage through lipid peroxidation; however, we determined that Candida SOD activity was activated in response to amphotericin B exposure. The mean SOD activity of the C. dubliniensis and C. albicans strains resistant to 2µg ml-1 amphotericin B was 1.97-fold greater than that of the sensitive strains. This SOD activation likely occurred due to the induction of an adaptive response upon exposure of Candida to sublethal concentrations of peroxide or superoxide. This adaptive response results in a several-fold increase in catalase, SOD, and glucose 6-phosphate dehydrogenase (G6PDH) activity29,31,32. In addition, the elevation of SOD activity in the yeast strains resistant to amphotericin B may contribute to an increased resistance to phagocytic cell attack, making the yeast cells more resistant to host defenses. We also observed that the SOD activities of the C. albicans strains that were sensitive to fluconazole and amphotericin B were significantly (p < 0.01) higher than the SOD activities of the sensitive C. dubliniensis strains. When SOD activity was compared among the resistant groups, C. albicans also expressed significantly (p < 0.01) higher levels of SOD than C. dubliniensis.

As previously demonstrated for other enzymes (e.g., proteinases), SOD activity was higher in C. albicans than in C. dubliniensis. This study is the first to evaluate the effect of fluconazole resistance on SOD activity in C. albicans and C. dubliniensis. Our results suggest that fluconazole has an oxidant effect on Candida, resulting in the activation of its antioxidant system, including SOD. The mean SOD activities of the fluconazole-resistant C. albicans and C. dubliniensis strains were 1.93- and 1.97-fold higher, respectively, than those of the sensitive strains. If fluconazole induces SOD activity, like amphotericin B, then this reaction to fluconazole would contribute to the increased resistance of Candida to phagocytic cell attack.

Similar to SOD activity, catalase activity was elevated in the fluconazole- and amphotericin B-resistant C. albicans and C. dubliniensis cells compared to the sensitive groups. The enzyme catalase converts hydrogen peroxide (H2O2) to oxygen (O2) and water (H2O)16. In Candida, catalase has been suggested to be involved in the resistance to amphotericin B and is involved in hypha formation15,28. Catalase activity has not been previously studied in fluconazole-resistant C. albicans and C. dubliniensis strains. Here, we demonstrated that the catalase activities of the amphotericin B-resistant C. dubliniensis and C. albicans strains were 2- and 1.77-fold higher, respectively, than those of the sensitive groups. The catalase activities of the fluconazole-resistant C. albicans and C. dubliniensis groups were 1.58- and 1.57-fold higher, respectively, than those of the sensitive groups. These findings demonstrate that similar changes in catalase and SOD activities occur in response to exposure to fluconazole and amphotericin B under the same conditions and corroborate the hypothesis that an adaptive response occurs when Candida is exposed to oxidative stress32. Based on the literature, we expected that these enzymes would have similar responses because the product of SOD is a substrate for catalase11. Here, we confirmed this enzymatic profile in C. albicans and C. dubliniensis. Similar to SOD, catalase activity was significantly (p < 0.01) higher in the sensitive and resistant C. albicans strains compared to the respective C. dubliniensis groups. In combination with the increased susceptibility of C. dubliniensis to hydrogen peroxide and macrophage killing, this result suggests that C. albicans is better protected from oxidative stress than C. dubliniensis, in accordance with the higher virulence of C. albicans33,34,35.

The results obtained in this study also demonstrate a synergism between SOD and catalase in the C. dubliniensis and C. albicans strains that were resistant to fluconazole and amphotericin B, in which protection by SOD generates hydrogen peroxide but not other ROS, such as lipid hydroperoxides or the reactive nitric intermediate (RNI) peroxynitrite, which would be more detrimental to the cells due to the absence of defenses against these types of compounds. Because the hydrogen peroxide generated by SOD activity is still toxic to the cell, it must be eliminated by catalase, which converts this toxic compound into water14.

In conclusion, this study is the first to determine the effect of fluconazole and amphotericin B resistance on catalase and SOD activities in C. albicans and C. dubliniensis. The stress-inducing effect of fluconazole on Candida has not been previously described, and we have demonstrated that catalase and SOD activities are increased in fluconazole-resistant cells. We also confirmed that C. albicans has more active antioxidant pathways than C. dubliniensis.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

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Received: September19, , 2013; Accepted: November26, , 2013

Address to: Dr. Carlos Eduardo Blanco Linares. Dept° Ciências da Saúde/URI. Av. Assis Brasil, 98400-000 Frederico Westphalen, RS, Brasil. Fax: [phone]. e-mail: [email protected]

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