Laboratory of Biophysics, School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea1
Author for correspondence: Sa-Ouk Kang. Tel: +82 2 880 6703. Fax: +82 2 888 4911. e-mail: kangsaou{at}plaza.snu.ac.kr
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ABSTRACT |
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Keywords: SOD1, hyphal growth, lysine auxotrophy, candidiasis
Abbreviations: Cu/ZnSOD, copper- and zinc-containing SOD; FeSOD, iron-containing SOD; MnSOD, manganese-containing SOD; ROS, reactive oxygen species; SG, synthetic glucose; SGU, SG plus 25µg uridine ml-1; SOD, superoxide dismutase
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INTRODUCTION |
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Superoxide dismutase (SOD), an anti-oxidant enzyme, catalyses the dismutation of superoxide radical anions to dioxygen and hydrogen peroxide. Generally, SODs are categorized into four classes according to their metal cofactors copper- and zinc-containing SOD (Cu/ZnSOD), manganese-containing SOD (MnSOD), iron-containing SOD (FeSOD) and nickel-containing SOD (Fridovich, 1989 , 1995
; Youn et al., 1996a
, b
). Cu/ZnSOD is found mostly in the cytosol (Fridovich, 1989
, 1995
) and mitochondria of eukaryotic cells (Okado-Matsumoto & Fridovich, 2001
) and in the periplasmic space of some prokaryotes (Battistoni et al., 1998
; Farrant et al., 1997
; Wilks et al., 1998
). This enzyme has been shown to play a role in protecting cells against oxygen toxicity (Fridovich, 1989
, 1995
) and to act as a major repository for copper ions in virtually all eukaryotes (Culotta et al., 1995
); however, the loss of Cu/ZnSOD activity has various pleiotropic consequences on organisms, which include slow growth, conditional auxotrophies and DNA damage (Fridovich, 1989
, 1995
). For example, a Cu/ZnSOD-null yeast strain was shown to be oxygen-sensitive, hypermutable, auxotrophic for lysine and methionine and defective in sporulation (Liu et al., 1992
). In some pathogenic organisms, Cu/ZnSOD has also been proposed as being a virulence determinant that could decompose the superoxide radical anions generated by phagocytic cells (Hong et al., 1992
; Farrant et al., 1997
; Wilks et al., 1998
; Battistoni et al., 1998
).
Candida albicans, the major fungal pathogen of humans, causes not only oral and vaginal thrush but also systemic or life-threatening infections in immunocompromised patients (Cutler, 1991 ; Coleman et al., 1993
). A number of factors are known to be involved in the virulence of C. albicans, such as its adhesion to host tissues, its evasion of the host immune system, its secretion of protease and its reversible morphological conversion from yeast to hyphal growth (Cutler, 1991
; Vázquez-Torres & Balish, 1997
). Once C. albicans has infected a host, it inevitably encounters ROS produced by host phagocytes as well as ROS produced as a consequence of its own oxygen metabolism. Since phagocytic cells produce the superoxide radical anion, the first intermediate in the sequential univalent reduction of dioxygen, during the oxidative burst, the SODs of C. albicans are thought to play a protective role in this organism by suppressing oxidative killing by the infected host (Vázquez-Torres & Balish, 1997
).
Although SODs are important anti-oxidant enzymes and have an additional hypothetical role in the virulence of pathogenic fungi (Hamilton & Holdman, 1999 ), there is no direct evidence that SODs are involved in the pathogenicity of C. albicans. We have previously reported the characterization of Cu/ZnSOD and its gene from C. albicans (Hwang et al., 1999
). The present study shows that Cu/ZnSOD of C. albicans contributes to the protection of this organism against oxidative stresses and to the establishment of the full virulence of this organism. This is the first report to provide direct evidence that Cu/ZnSOD is involved in the virulence of pathogenic fungi.
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METHODS |
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Disruption and re-integration of C. albicans SOD1.
To disrupt both alleles of SOD1 using the URA-blaster technique (Fonzi & Irwin, 1993 ), the oligonucleotide primers 5'-GTTCATTTTGAACAAGAA-3' and 5'-GCCAATGACACCACAAGC-3' were used. The resulting 402 bp PCR product was inserted into the pGEM-T Easy vector (Promega), yielding pCH101. A 4·1 kb hisGURA3hisG cassette from p5921 (Fonzi & Irwin, 1993
) was then inserted into a portion of SOD1 within pCH101. The resulting plasmid, pCH102, was cut with ApaI/SacI, to remove the vector, and the disrupted SOD1 gene was transformed into the Ura- C. albicans strain CAI4 (Fonzi & Irwin, 1993
). Ura+ transformants were selected on an uracil-deficient medium and the integration of the hisGURA3hisG cassette into the SOD1 locus was verified by Southern blot analysis. Spontaneous Ura- derivatives of the heterozygous disruptants were selected on SG medium containing 625 µg 5-fluoroorotic acid ml-1 and 100 µg uridine ml-1. This procedure was repeated to delete the remaining functional allele of SOD1.
For re-integration of SOD1 into the genome of C. albicans, an XbaI/ScaI-digested URA3 fragment from pURA3 (Huh et al., 2001 ) was inserted into the XbaI/EcoRV sites of pSOD1, which was constructed through the ligation of a 2·8 kb EcoRI-digested genomic DNA fragment containing the SOD1 coding region into pBluescript KS(+) (Stratagene). The resulting plasmid, pCH103, was linearized by digestion at the unique HpaI site upstream of the coding region of SOD1; the linearized plasmid was integrated into the SOD1 locus of the Ura- strain sod1/sod1. The targeted re-integration of SOD1 into the genome of sod1/sod1 was confirmed by PCR (data not shown) and Southern blot analysis.
Staining to detect SOD activity.
SOD activity on a non-denaturing polyacrylamide gel was detected by negative staining (Manchenko, 1994 ). The gel was incubated in 50 mM phosphate buffer (pH 7·8) for 10 min, in nitro blue tetrazolium solution (1 mg ml-1) for 10 min and then in 50 mM phosphate buffer (pH 7·8) containing 0·01 mg riboflavin ml-1 and 3·25 mg N,N,N',N'-tetramethylethylenediamine ml-1 for 10 min at room temperature with gentle shaking. Areas of SOD activity remained clear when the gel was exposed to the light.
Western blot analysis.
The purified Cu/ZnSOD enzyme (Hwang et al., 1999 ) was isolated from a 12% SDS-polyacrylamide gel and then injected into a mouse (4-week-old ICR female). Boosting with purified Cu/ZnSOD was done twice, with a 2-week interval between each boost; the mouse was killed 10 days after the second boost. A total of 50 µg of cell-free extract of C. albicans was subjected to 12% SDS-PAGE. The protein was then transferred onto a nitrocellulose filter and electroblotting was done according to Bollag & Edelstein (1991)
. The signals were visualized by using a colorimetric detection kit (Roche Molecular Biochemicals), according to manufacturers instructions.
Determination of growth rate.
To assess the yeast-phase growth rates, an overnight-grown culture of C. albicans was subcultured into YPG or SG media and incubated at 28 °C. The cell density (OD600) of these cultures was measured after 4 h and then every 2 h up to the stationary phase. The doubling time of the cultures during the exponential phase was determined by using the formula ln2xt/(lnb-lna), where t is the time period in hours, a is the cell density at the beginning of the time period and b is the cell density at the end of the time period. To determine the extension rate of the hyphae, C. albicans cells were incubated in YPG containing 10% fetal bovine serum (FBS; Gibco-BRL) at 37 °C and the relative sizes of the C. albicans hyphae were measured every 1 h over a period of 4 h under a microscope.
Assay for the resistance of C. albicans to oxidative stresses.
All experiments were done according to the method of Izawa et al. (1995) with some modifications. Cells were grown to mid-exponential phase (2x107 cells ml-1), harvested and resuspended in 50 mM potassium phosphate buffer (pH 7·8), to obtain an initial OD600 value of 0·1. To observe the sensitivity of the C. albicans cells to oxidants, various concentrations of menadione or hydrogen peroxide were added to the cell suspensions. After 1 h incubation at 28 °C, aliquots were taken from the cell suspensions, diluted appropriately in 50 mM potassium phosphate buffer (pH 7·8) and plated onto solid YPG medium. After 2 days incubation at 28 °C, the number of colonies on the plates was counted.
Survival assay in macrophages.
To determine the survival rate of C. albicans cells exposed to macrophages (cell-line RAW264.7), we used an end-point dilution survival assay (Rocha et al., 2001 ). An aliquot (1 ml) of an overnight-grown culture of C. albicans cells was washed twice in PBS and resuspended at 1x107 cells ml-1 in cold 10% heat-inactivated FBS. An aliquot of the suspension (50 µl) was added to 150 µl cold 10% heat-inactivated FBS in 96-well plates containing medium or only macrophages (1x105 cells per well). After four-fold serial dilutions, the plates were incubated on ice for 30 min and subsequently for 24 h at 37 °C in 5% CO2. Colonies were visualized with the use of an inverted-phase microscope (Zeiss Axiovert 100) at 40x magnification. Survival of C. albicans was expressed as a percentage of the number of colonies in the presence of macrophages divided by the number of colonies in the absence of macrophages. Data shown represent the mean±SE of three independent experiments.
Virulence studies.
Inbred BALB/c mice (Seoul National University Laboratory Animal Center) weighing between 17 and 20 g were used to test the virulence of the different strains of C. albicans. Two experiments were initiated by growing the C. albicans strains on YPG plates for 48 h at 28 °C, suspending the cells in PBS and adjusting the suspensions to the desired cell density (OD600=0·5). The virulence of each C. albicans strain (i.e. SC5314, CH103 or CH104R) was tested by injecting 0·1 ml of the appropriate cell suspension (1x106 cells) into five mice. The statistical analyses of the differences in survival between the paired groups were performed with the KaplanMeier log-rank test. A P value of 0·05 was taken to indicate statistical significance in the results.
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RESULTS |
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To examine the effect of the sod1 mutation on the hyphal growth of C. albicans, Ura+ prototrophs were grown on several solid or liquid media that induce hyphal growth, e.g. Spider medium (Liu et al., 1994 ), Lees medium (Lee et al., 1975
), 10% serum, corn meal agar (Difco) containing 0·33% Tween 80 and RPMI 1640 medium (Gibco-BRL). Although closer examination of the sod1/sod1 strain revealed no clear differences in the morphological phenotypes of this strain when grown in/on the different media tested, the sod1/sod1 mutant did show significantly delayed hyphal growth on solid Spider medium. The sod1/sod1 mutant had a wrinkled colony phenotype when grown on Spider medium for 5 days at 37 °C, whereas wild-type and heterozygote cells formed extensive radial filaments when grown on this medium (Fig. 3
). When grown on Spider medium the revertant strain (CH104R) regained the capacity to set up hyphal growth, consistent with that of the heterozygote strain grown under the same conditions (Fig. 3
).
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Virulence study
To investigate the effect of the sod1 defect on the virulence of C. albicans in a BALB/c mouse model, wild-type, sod1/sod1 and revertant strains of C. albicans were intravenously injected into immunocompetent mice. Since the Ura- strains show decreased virulence, only isogenic Ura+ strains were used to infect the mice. All of the mice inoculated with the wild-type or revertant cells carrying SOD1 died within 9 days of being inoculated. However, the mice inoculated with the Cu/ZnSOD-deficient strain survived significantly longer than those inoculated with the wild-type parental or revertant strains in each of two separate experiments (Fig. 7). The survival differences between the sod1/sod1 and wild-type or revertant strains were significant (P<0·05, according to the KaplanMeier log-rank test). This result indicated that Cu/ZnSOD contributes to the virulence of C. albicans in a mouse model of intravenous infection.
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DISCUSSION |
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C. albicans sod1/sod1 cells had similar phenotypes to S. cerevisiae sod1 cells with respect to their sensitivity to increasing concentrations of menadione (Fig. 4), their slow aerobic growth in minimal medium (Table 2
) and their leaky lysine auxotrophy (Fig. 6
). However, the sod1/sod1 mutant showed no auxotrophy for methionine or cysteine. The requirement of either methionine or cysteine to be present for the growth of S. cerevisiae sod1 cells is apparently not a true auxotrophy, but rather results from the metabolic consequences of sulfur assimilation, which increases in the absence of sulfur-containing amino acids (Chang et al., 1990
). Thus, the C. albicans sod1/sod1 cells examined here seem to be more resistant to attack by the superoxide radical anion-mediated sulfur radicals formed during sulfur assimilation than S. cerevisiae sod1 cells. Recently, it has been reported that C. albicans has an unusual cytoplasmic MnSOD that confers anti-oxidant protection to the organism during its growth phases (Lamarre et al., 2001
); this cytoplasmic MnSOD has also been found in a few other organisms, including unicellular green algae (Kitayama & Togasaki, 1995
) and filamentous fungi (Diez et al., 1998
). Unlike the situation with the S. cerevisiae sod1 strain, the cytosolic MnSOD of C. albicans, possibly, relieves the detrimental effect of a sod1 defect in this organism. Therefore, it is plausible (as one of a number of explanations) that the C. albicans sod1/sod1 cells have a diminished requirement for the essential amino acids lysine, methionine or cysteine to be present in minimal medium when grown under aerobic conditions compared to that of S. cerevisiae sod1 cells. An N. crassa sod1 mutant did not show any auxotrophy when grown aerobically in minimal medium (Chary et al., 1994
). Taken together, these results indicate that across fungal species there might be, to varying extents, differences in the abilities of different species to resist toxic products derived from superoxide radical anions.
C. albicans is a member of the normal microflora of humans and does not usually cause disease in immunocompetent hosts. However, C. albicans causes serious diseases in immunocompromised individuals, such as patients suffering from leukaemia or diabetes, those that have undergone recent organ transplant and human-immunodeficiency-virus-infected individuals (Coleman et al., 1993 ). For the removal of C. albicans from the infected host, infected host cells require the interaction of many different types of immune cells with several candidacidal mechanisms. Oxygen-dependent killing mechanisms are very important in the removal of C. albicans from the infected host; these include the superoxide radical anion, myeloperoxidase hydrogen peroxide/halide system and reactive nitrogen intermediate responses of host macrophages (Vázquez-Torres & Balish, 1997
). Therefore, the anti-oxidant defence systems of C. albicans are assumed to be essential for this organism to resist the host immune response and for it to exhibit its full virulence. In agreement with this view, exogenous anti-oxidants have been shown to impair the killing of C. albicans cells by neutrophils (Wagner et al., 1986
) and a catalase-deficient (Wysong et al., 1998
) or erythroascorbic acid-deficient C. albicans strain (Huh et al., 2001
) has been shown to be far less virulent for mice than the parental wild-type strain. The present study also shows that the Cu/ZnSOD-deficient strain used here has an increased susceptibility to fungicidal damage by macrophages and attenuated virulence in a mouse model for systemic candidiasis (Fig. 7
). Considering the function of Cu/ZnSOD as an anti-oxidant enzyme, our results and those of others suggest that Cu/ZnSOD may be essential for C. albicans to resist the oxidant-mediated killing actions of the host immune system. Another possible explanation for the attenuated virulence of the C. albicans sod1/sod1 cells observed here is likely to involve lysine biosynthesis. Since host cells do not possess the ability to synthesize lysine, an essential nutrient, infecting C. albicans cells can only utilize this limited nutrient via a de novo pathway (Broquist, 1971
). However, this possibility seems to be very unlikely because the differences, if any, in the doubling times and yields between sod1/sod1 and wild-type or revertant pathogenic Ura+ strains were very small when the strains were grown in minimal medium (Table 2
). It is also well known that there is a strong correlation between the morphological transition of C. albicans from yeast-like to hyphal growth and its virulence. The positive control of hyphal development in C. albicans is signalled, to a certain extent, via the MAP kinase cascade to activate the transcription factor Cph1, whose deletion results in suppressed hyphal growth of C. albicans on Spider medium (Liu et al., 1994
). The cph1/cph1 mutant can develop hyphae when grown in serum or liquid media and is as virulent as the wild-type strain (Lo et al., 1997
). Since C. albicans sod1/sod1 cells still form hyphae when grown in liquid culture and in response to serum (like the cph1/cph1 mutant cells), the delayed hyphal growth of this strain on Spider medium is unlikely to be the major cause of its attenuated virulence.
In conclusion, we have shown that Cu/ZnSOD of C. albicans plays roles in the protection of this organism from oxidative stresses, in its defective hyphal development on Spider medium, in its survival in macrophages and in the establishment of its full virulence in a mouse infection model. Thus, our findings demonstrate that Cu/ZnSOD is involved in the virulence of C. albicans and provide important clues as to how the anti-oxidant enzymes of fungal pathogens function in the infection process.
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ACKNOWLEDGEMENTS |
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Received 26 March 2002;
revised 22 May 2002;
accepted 18 July 2002.