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Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA
Correspondence
David T. Kirkpatrick
dkirkpat{at}umn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). However, it can cause significant mortality in immunocompromised individuals or patients whose microflora has been altered. The mortality rate associated with systemic candidiasis approaches 35 %, even with antifungal drug treatment (Pittet & Wenzel, 1995). Although C. albicans is closely related to the model yeast Saccharomyces cerevisiae, C. albicans is the fourth most common cause of nosocomial (hospital-acquired) infections, while S. cerevisiae is considered a non-pathogenic yeast. Therefore, comparisons between these two fungi have proven to be useful in understanding the pathogenicity of C. albicans.
Several reports have shown karyotypic diversity in clinical isolates of C. albicans and in laboratory strains grown under stress (Janbon et al., 1998; Magee et al., 1992). Studies have demonstrated that gross chromosome rearrangements, such as chromosome gain/loss or isochromosome formation, occur in clinical isolates that have become drug resistant (Legrand et al., 2004; Selmecki et al., 2006). In addition, chromosome loss or gain has been observed in laboratory strains that became resistant to antifungal drugs or acquired the ability to utilize sorbose as the sole source of carbon (Janbon et al., 1998; Perepnikhatka et al., 1999). In these cases, a change in chromosome number correlates with the appearance of a new advantageous phenotypic trait.
The nucleotide excision repair (NER) pathway is the primary repair pathway for removal of DNA-distorting lesions such as UV light-induced thymine dimers. In S. cerevisiae, NER is mediated by multiple proteins. Together Rad1p and Rad10p form a single-stranded DNA endonuclease that binds DNA and then nicks the damaged DNA strand on the 5' side of the lesion (Tomkinson et al., 1993). Rad2p is a separate endonuclease that cleaves 3' of the site of damage (Habraken et al., 1993). The action of these endonucleases releases the single-stranded DNA containing the lesion. Subsequent DNA repair synthesis fills in the 
25 nt gap. The Rad1/10 protein complex also removes non-homologous tails during mitotic recombination (Ivanov & Haber, 1995), acts during meiotic gene conversion of large single-stranded loops in heteroduplex DNA (Kirkpatrick & Petes, 1997), and when mutated results in an elevated level of chromosome gain or loss (Howlett & Schiestl, 2004; Murray et al., 1994; Sun et al., 2003).
The base excision repair (BER) pathway removes lesions that result from exposure to endogenous or exogenous reactive oxygen species (ROS) (reviewed by Memisoglu & Samson, 2000). BER takes place in two steps. In S. cerevisiae, a glycosylase (e.g. Ntg1p, Ntg2p and Ogg1p, among others) removes the damaged base, creating an abasic (AP) site, which is a substrate for an AP endonuclease (Apn1p or Apn2p). The AP endonuclease cleaves the DNA backbone, forming a single-nucleotide gap, allowing DNA synthesis to replace the missing base. There is significant functional redundancy in BER, with multiple glycosylases recognizing different lesions. In addition, studies have shown an overlap between the NER and the BER pathways in S. cerevisiae. For instance, Torres-Ramos et al. (2000) demonstrated that the NER complex competes with Apn1p and Apn2p in the repair of AP sites.
Base or nucleotide excision repair may play a role in the development of systemic candidiasis, which requires survival of C. albicans cells upon ingestion by macrophages, the first line of defence against C. albicans infection. Macrophages kill microbes by producing a huge amount of ROS molecules (also called an oxidative burst), leading to the possibility that BER or NER might be important in Candida infection.
To investigate the roles of the BER and NER pathways in C. albicans in order to determine their effects on mutation, chromosomal instability and acquisition of antifungal drug resistance, we constructed disruptions in genes already shown in S. cerevisiae to be central components of BER and NER and tested them for DNA repair defects. We observed that mutants defective in the BER pathway are not sensitive to oxidizing agents, nor are they altered in their response to macrophages or various antifungal drugs. Mutants defective in the NER pathway are very sensitive to UV radiation, but do not exhibit an altered response to antifungal drugs. Mutation rate and chromosome 1 (Ch1) integrity were monitored using a GAL1/URA3 system, and no significant difference was observed between the DNA repair mutants and the parental strain. Our data suggest that redundancy within each pathway and between pathways is likely to be more pronounced in C. albicans than in S. cerevisiae. Our data also showed that there are differences between the S. cerevisiae and C. albicans NER and BER pathways, which may shed some light on the different lifestyles of these two fungi.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. C. albicans and S. cerevisiae strains were maintained on YEPD media (1 % yeast extract, 2 % peptone, 2 % glucose) supplemented with 20 mg uridine l–1 at 30 °C. Construction of the parental strain DKCa39 has been described previously (Legrand et al., 2007). The S. cerevisiae strains used in this study are diploid derivatives of BY4743 (Brachmann et al., 1998), which is MATa/
his3
1/his31 leu20/leu20 lys20/LYS2 MET15/met150 ura30/ura30. All derivative strains homozygous for deletions in NTG1 (SCD1), APN1 (SCD2), RAD2 (SCD4), RAD10 (SCD5) or OGG1 (SCD142) were obtained from the Saccharomyces Genome Deletion Consortium strain set (Winzeler et al., 1999). Names in parentheses (e.g. SCD1) are internal reference names only.
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). The first allele was replaced with the Candida dubliniensis HIS1 marker and the second with the C. albicans or C. dubliniensis ARG4 marker. The cassettes were amplified with the same set of primers, since the HIS1 and ARG4 markers have been cloned into the same location in the same vector (Noble & Johnson, 2005). Transformations used a variation of the standard lithium acetate transformation protocol (Legrand et al., 2007). Transformants were initially screened by colony PCR with primers positioned within the marker sequence and outside the integration site. Positive transformants were cultured for DNA extraction, and both boundaries of the integration site were verified by PCR. For each gene, two independent homozygous diploid mutants were independently constructed. Reintegration strains were constructed by integrating a wild-type copy of the appropriate gene at its endogenous locus with the NAT1–FLP cassette (Shen et al., 2005), using protocols described previously (Legrand et al., 2007). Because the reintegration cassette replaces either the HIS1 or the ARG4 deletion marker, positive transformants become auxotrophic for either histidine or arginine, thereby indicating the chromosome homologue bearing the reintegration construct. Reintegration strains were confirmed by PCR (Legrand et al., 2007).
Phenotype assays.
Growth rate determination was done using protocols described previously (Legrand et al., 2007). Doubling times were determined in two independent experiments using the following formula:
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Colony and cell morphologies, chlamydospore formation and filamentation were analysed as described previously. Cells were examined using a Nikon E600 microscope to determine cell morphology, filamentation and chlamydospore formation. Pictures of colonies were obtained with a Nikon CoolPIX900 camera attached to a Zeiss Stemi DRC microscope.
Antifungal drug resistance was monitored using E-test strips (AB Biodisk) on casitone+uridine agar plates (0.5 % yeast extract, 1 % sodium citrate, 0.9 % Bacto casitone, 2 % glucose, 2 % agar, 20 mg uridine l–1), as described previously (Legrand et al., 2007).
DNA damaging agent sensitivity.
All assays were conducted as described previously (Legrand et al., 2007). For sensitivity to oxidizing agents, YEPD+uridine plates containing 4 mM H2O2, 0.1 mM menadione or 2 mM tetrabutyl hydrogen peroxide (TBHP), or 5 ml YEPD+uridine+4 mM H2O2 liquid culture media were used. For alkylating-agent sensitivity, cells were placed on YEPD+uridine plates containing 100 µM camptothecin, 0.03 % ethylmethane sulfonate (EMS) or 0.01 % methylmethane sulfonate (MMS) and incubated at 30 °C for 24 h. For UV radiation sensitivity, appropriate dilutions of cells on plates were irradiated with 3.2 µW cm–2 UV light for 5 s. To quantify survival upon UV irradiation, 107 cells per strain were put on solid media and exposed to 7.0 µW cm–2 of UV for 3 or 5 s. Plates were incubated in the dark for 48 h at 30 °C and colonies were counted. To obtain a background level of sensitivity, 300 cells of the parental wild-type strain DKCa39 were plated and subjected to the same UV treatment as the other plates.
Ch1 integrity.
All protocols were performed as described previously (Legrand et al., 2007). Cells (100 000) were plated on YEPD+uridine plates, and minimal+2-deoxygalactose (2-DG) and minimal+5-fluoroorotic acid (5-FOA) plates. The number of colonies on each plate was recorded on day 2 for the YEPD+uridine plates and on day 3 for the 2-DG and 5-FOA plates. To characterize the spectrum of alterations leading to 2-DG and 5-FOA resistance, 20 2-DGR and 20 5-FOAR colonies were selected, and genomic DNA was extracted and screened by PCR to assess the presence/absence of the GAL1 and URA3 genes. The oligonucleotides CaGAL1+474-F, CaGAL1-256-R and CaURA3+386-R were used in the same PCR mix (Legrand et al., 2007). If the GAL1 or URA3 genes were still present in 2-DGR or 5-FOAR cells, respectively, the PCR product was sequenced, while if they were absent, single nucleotide polymorphism (SNP) analysis was used to determine the extent of the loss. SNPs 1322/2294 and F12n4 are located on Ch1, flanking the GAL1/URA3 locus; both SNP sequences contain a restriction site polymorphism (Fig. 4). The regions containing the SNPs were amplified by PCR using the oligonucleotides Ca1322-2294-Chr1SNP-F/R and CaF12n4-Chr1SNP-F/R (Legrand et al., 2007). The PCR product was digested with either BccI or HpaII, respectively, and analysed by gel electrophoresis.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), suggesting that the C. albicans genes are likely to be orthologues of the S. cerevisiae DNA repair genes.
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). At this point, it is not clear to us why the BLAST search did not detect the NTG2 orthologue at the nucleotide level.
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/ ntg1/ and ogg1/ ntg1/ double mutants.
The colony morphologies of the BER and NER mutants did not differ from that of wild-type colonies. In contrast to the colony and cell morphology phenotypes exhibited by double-strand-break repair mutants (Andaluz et al., 2006; Legrand et al., 2007), including strains derived from the same parental background as used in this study, the mutants grew like wild-type yeast cells in liquid cultures at 30 °C (data not shown). We characterized the growth rates of the deletion mutant strains and observed that the BER and NER mutants had a doubling time similar to that of the parental strain (Table 4).
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). Based on these data, we investigated filamentation in the BER and NER mutants, but did not observe any morphogenesis defects. The parental strain and the DNA repair mutants grew as yeast cells in YEPD+uridine at 30 °C and formed germ tubes when grown in YEPD+uridine+10 % serum and Spider media at 37 °C (data not shown).
We investigated another morphological characteristic of C. albicans, its ability to produce large thick-walled spores called chlamydospores, which are induced upon oxygen limitation. Hog1p, a mitogen-activated protein (MAP) kinase activated in response to oxidative stress (Alonso-Monge et al., 1999), is required for oxidative stress response and chlamydospore formation (Alonso-Monge et al., 2003). These observations led us to examine chlamydospore formation in the BER and NER mutants; all were capable of producing chlamydospores. There was no delay in the appearance of the chlamydospores and no difference in their frequency or appearance (data not shown).
Susceptibility to DNA-damaging agents
In many organisms, different DNA repair mutants exhibit different susceptibilities to various types of DNA-damaging agents. We tested the sensitivity of the BER and NER mutants to numerous agents such as UV radiation, oxidizing agents and alkylating agents, including compounds known to induce double-strand breaks.
We observed that the strains that contained homozygous deletions of the NER genes (rad2/ and rad10/) were extremely sensitive to UV light (at least 10 000-fold) whereas the BER mutants did not show any change in their UV sensitivity as compared with the parental strain (Fig. 2). Reintegration strains were constructed to confirm the phenotypes observed in the rad2/ and rad10/ mutants. The UV-sensitivity phenotype was rescued in the reintegration strains (Fig. 2). We further characterized UV sensitivity by determining survival percentages upon UV irradiation (Table 5). In the wild-type parental strain, 73 % of the cells were able to form colonies after exposure to 3 s of UV; this percentage was reduced to 66 % after 5 s of exposure. In contrast, less than 0.01 % of the rad10 or rad2 cells survived after 3 s of exposure, with a further 10-fold reduction after 5 s. Considerable variability in survival was seen in the mutant strains, as demonstrated by the large SDs observed (Table 5), but all mutants exhibited at least a four orders of magnitude loss of viability following very brief exposure to UV radiation. This extreme sensitivity of the NER mutants to UV radiation is consistent with the role that the NER pathway plays in repairing UV light-induced thymine dimers.
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concluded that C. albicans does not possess a photoreactivating enzyme. We searched the genome sequence of C. albicans for photolyase homologues and did not find any good candidates, in agreement with an earlier report of a BLAST search of the C. albicans genome (Ciudad et al., 2004). However, a photoreactivation activity could be masked by a very efficient NER pathway, which would be uncovered by the NER mutants. We compared the UV sensitivity of NER mutant strains incubated in the dark to that of the same strains exposed to visible light following UV irradiation. We did not observe any difference (data not shown), further strengthening the conclusion of Miller & Sarachek (1974) that C. albicans lacks a photoreactivating enzyme.
We noted that the level of UV sensitivity of the C. albicans rad2/ and rad10/ mutants was higher than that of the S. cerevisiae rad2/ and rad10/ mutants (Fig. 2
, Table 5). In addition, the S. cerevisiae mutants appeared to recover more readily after UV treatment as compared with the C. albicans mutants (Fig. 2). These differences could result from the specific environmental conditions in which the two yeasts grow. S. cerevisiae is commonly found on leaf and fruit surfaces, where the cells would be directly exposed to the UV light found in sunlight, whereas the normal habitat of C. albicans is the mucosal membranes of humans and other warm-blooded animals, and therefore it would be unlikely to be exposed to significant amounts of UV light. Because of its high level of exposure, S. cerevisiae might have developed back-up pathways capable of repairing UV-induced damage, or there might be more overlap between different DNA repair pathways. These back-up pathways might be absent in C. albicans, or there might be less overlap between the different DNA repair pathways, explaining why C. albicans mutants with defects in NER do not recover after UV irradiation as well as comparable S. cerevisiae mutants.
We tested the sensitivity of the C. albicans and S. cerevisiae BER and NER mutants to the DNA break-inducing agents camptothecin, EMS and MMS. The C. albicans BER and NER mutants exhibited a wild-type level of sensitivity to all of the compounds (data not shown), demonstrating that BER and NER do not play a major role in repairing DNA breaks. This result is in contrast to C. albicans double-strand-break repair mutants, as we have shown previously that these mutants exhibit susceptibility to camptothecin, EMS and MMS (Legrand et al., 2007).
We tested the response to two oxidizing agents, H2O2 and TBHP, using a plate-based assay. Our data did not show any difference in the sensitivity to oxidizing agents between the mutant strains and the parental strains (Fig. 3). In S. cerevisiae, multiple gene disruptions are required in order to observe deficiencies in BER; for some genes, components of the NER pathway must also be disrupted. Our data suggest that BER in C. albicans may have similar levels of redundancy. An interesting observation was that C. albicans appears to be more resistant to TBHP and H2O2 than S. cerevisiae (Fig. 3). This observation could reflect the adaptation of C. albicans to its pathogenic lifestyle, as the first line of defence during C. albicans infection is macrophages, which kill yeast cells by oxidative burst (production of ROS resulting in damage to the DNA molecules). In order to escape, C. albicans must be able to deal very efficiently with macrophage-induced oxidative damage during an immune response. Any cells that survive must also deal with ROS-induced DNA damage, perhaps explaining why the BER pathway of C. albicans is much more efficient than that of S. cerevisiae.
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). The same property could contribute to the ability of C. albicans cells to survive within macrophages and even escape from macrophages. We used C. albicans DNA repair mutants to evaluate the role of BER in C. albicans pathogenesis and to determine whether one reason for C. albicans survival in macrophages is the repair of DNA bases oxidized by macrophage oxidants. To do this, we incubated the C. albicans cells (wild-type or mutants) with J774A murine macrophage-like cells. After 3 h, we measured survival of C. albicans cells using an XTT colorimetric method (Sigma; Meshulam et al., 1995). Within the limits of our assay, the ntg1 and apn1 mutations did not affect fitness after phagocytosis (data not shown). One possible follow-up for this assay is to disrupt APN1 or NTG1 in cells that do not filament. Such cells become trapped in macrophages for extended times (up to 48 h), allowing observation of more subtle phenotypes due to prolonged exposure. If macrophages were causing DNA damage of the kinds that one would expect from oxidizing agents, the effect of that damage might be manifested over a longer time frame than the one used in this assay.
Chromosome instability assays
We investigated mutation and the maintenance of Ch1 stability with a GAL1/URA3 marker system. One allele of the GAL1 locus, located on Ch1, was replaced with a copy of the URA3 gene, allowing us to readily distinguish between the two Ch1 homologues. As this was done in the parental strain, the genotype of all of the mutant derivatives is GAL1/gal1 : : URA3. The integrity of Ch1 was assessed by growing strains on media containing either 2-DG, which kills GAL1+ cells, or 5-FOA, which kills URA3+ cells. Resistance to 2-DG or 5-FOA could arise from mutation of the genes or their loss through gene conversion, interstitial deletion or aneuploidy.
The frequency of appearance of 2-DGR and 5-FOAR colonies was compared in the parental strain and the DNA repair mutant strains. We observed a two- to threefold increase in 2-DGR colonies in the NTG1, RAD10 and RAD2 mutants compared with the parental strain, but this was not accompanied by a similar increase in 5-FOAR colonies (Table 6). Conversely, a ninefold decrease in 5-FOAR colony frequency was detected for the OGG1 mutant, while there was no change in the frequency of 2-DGR colonies. These results contrast with those of our previous study, in which MRE11 and RAD50 mutants exhibited eight- to 16-fold increases in resistant colonies for both GAL1 and URA3 loci. We do not consider these frequency alterations to be significant, given the small degree of change (except in the OGG1 mutant) and the observed bias in loss, especially when compared with the phenotypes of the DSBR mutants. The locus specificity seen with the NER and BER mutants may reflect a bias with respect to which homologue is mutated. In agreement with this, there was a twofold difference in alteration frequency between GAL1 and URA3 in the wild-type parental strain (Table 6). The alterations in mutation frequency do not result from an increase in mutations within the GAL1 or URA3 gene (see below), indicating that their primary sequences are unlikely to be the cause of the alterations in observed mutation frequency.
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SNPs located on both sides of GAL1/URA3 were then used to distinguish between gene conversion, segmental/total chromosome loss, break-induced replication (BIR) and reciprocal crossover. Flanking heterozygous SNPs in the 2-DGR or 5-FOAR strains would indicate that the cells lost GAL1 or URA3 by localized gene conversion; homozygosity would suggest that the cells underwent full-length chromosome loss, and if one of the SNPs remained heterozygous while the other became homozygous, some form of partial chromosomal deletion had likely occurred. The 1322/2294 SNP is located near the telomere of Ch1, while the F12n4 SNP is located close to the putative centromere of Ch1, but on the opposite side from the GAL1 locus (Fig. 4). When we characterized 2-DGR and 5-FOAR isolates of the wild-type parental strain previously, SNP typing showed that the telomere-proximal 1322/2294 SNP consistently became homozygous for the same allele, while the F12n4 SNP remained heterozygous, indicating that a large portion of the left arm of Ch1 had undergone a loss of heterozygosity (LOH) event (Legrand et al., 2007). The characterization of the 2-DGR and 5-FOAR colonies in the BER and NER mutants showed a similar pattern: all of the resistant isolates had lost the gene, and also become homozygous for the 1322/2294 SNP, while retaining heterozygosity of F12n4. Because the GAL1/URA3 locus is located 450 kb away from the telomere, it is more likely that LOH results from either reciprocal cross-overs or BIR in which one chromosomal arm is duplicated using the homologue as a template. We did not detect any alterations at the GAL1/URA3 locus in the NER mutants that would be consistent with an increase in chromosome nondisjunction or mitotic gene conversion errors. This result indicates either that the C. albicans NER proteins do not have a role in these events, unlike their counterparts in S. cerevisiae and Schizosaccharomyces pombe, or that the events occur too infrequently in C. albicans to be detected by our assay system.
Antifungal drug resistance
To determine the roles that the DNA repair genes may play in the acquisition of antifungal drug resistance, we tested the susceptibility of BER and NER mutants to fluconazole, using an E-test assay. The MIC values for the BER and NER mutants were determined after 48 h of exposure. We observed that the apn1/, ntg1/, ogg1/, rad2/ and rad10/ single mutants, as well as the apn1/ ntg1/ and ogg1/ ntg1/ double mutants, had a MIC of 0.38 µg ml–1, identical to that of the parental strain. We have shown previously that loss of mismatch repair or loss of double-strand-break repair leads to an increase in the appearance of fluconazole-resistant colonies within the inhibition ellipse (Legrand et al., 2007). We did not observe drug-resistant colonies for the BER and NER mutants, indicating that these pathways do not play a major role in generating the types of genome changes that correlate with the acquisition of drug resistance in C. albicans.
In conclusion, we have shown that strains that are defective in the NER pathway, the primary repair pathway for removal of UV light-induced thymine dimers, are extremely sensitive to UV light. We also observed that the BER mutants, in comparison with the parental strain, do not show any change in their sensitivity to DNA-damaging agents. Functional redundancy within the BER pathway, as well as an overlap with other DNA repair pathways, could explain the resistance of the BER mutants to oxidizing agents. Overall, we show that although the BER and NER pathways do not appear to play a major role in genome stability and antifungal drug sensitivity, interesting differences between C. albicans and S. cerevisiae shed some light on the extent and nature of divergence between these two fungi.
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ACKNOWLEDGEMENTS |
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Edited by: K. Kuchler
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 15 February 2008;
revised 20 May 2008;
accepted 26 May 2008.
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