Spindle assembly checkpoint component ... - Wiley Online Library

Molecular Microbiology (2002) 45(1), 31–44

Spindle assembly checkpoint component CaMad2p is indispensable for Candida albicans survival and virulence in mice Chen Bai,† Narendrakumar Ramanan,† Yan Ming Wang and Yue Wang* Microbial Collection and Screening Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609. Summary Here, we report an indispensable role for spindle assembly checkpoint (SAC) component CaMad2p in the survival and virulence of Candida albicans in mice. We hypothesized that cell cycle checkpoint functions, especially those monitoring the integrity of DNA and chromosome segregation, might be required for the pathogen to repair damage caused by host defence. To test this idea, we created SACdefective mutants by deleting the CaMAD2 gene that encodes a key component of the SAC pathway. The CaMAD2 mutant appears normal in morphology, growth rate and growth mode switch in unperturbed conditions. However, it quickly loses viability when treated with nocodazole, which causes disassembly of mitotic spindles. The mutant also exhibits increased frequency of chromosome loss. The virulence of the mutant is greatly reduced in mice, presumably because of the inability of the mutant cells to stop the cell cycle when the host defence damages cellular components important for chromosome segregation. Supporting this hypothesis, unlike the wild-type cells that can proliferate within and eventually grow out of macrophages, most of the CaMAD2 null mutant cells are unable to survive. This study suggests that SAC is required for survival of C. albicans in the host and could thus be targeted for anti-C. albicans therapies. Introduction The innate antimicrobial defence system of mammals is armed with a wide range of powerful weaponries, such as reactive oxygen and nitrogen intermediates (Nathan and

Accepted 26 March, 2002. *For correspondence. E-mail mcbwangy@ imcb.nus.edu.sg; Tel. (+65) 6 778 3207; Fax (+65) 6 779 1117. †These authors contributed equally to this work.

© 2002 Blackwell Science Ltd

Shiloh, 2000; Vazquez-Torres and Fang, 2001), antibiotic peptides (Stenger et al., 1998), lysosomal enzymes and growth inhibitors (Hiemstra et al., 1993; Vidal et al., 1993). To establish infection, microbial pathogens have evolved various strategies to evade, circumvent or subvert the host defence mechanisms. For example, many microbial cells can survive and proliferate within phagocytic cells using one or more of the following means: blocking phagosome–lysosome fusion (Gordon et al., 1980), preventing acidification of phagolysosomes (Eissenberg et al., 1993), increasing the production of superoxide dismutase (De Groote et al., 1997), breaking the phagosome membranes (Lo et al., 1997) or synthesizing a thick protective capsule (Daffe and Etienne, 1999). Despite these counteractive strategies in the battle against host defence mechanisms, damage to many key cellular components of the pathogens, such as DNA and proteins, is inevitable and must be repaired for survival. Eukaryotic organisms use cell cycle checkpoints to monitor the integrity of DNA and cellular structures that carry out accurate chromosome segregation during cell division (Hartwell and Weinert, 1989). Upon detecting damage or defects in these cellular structures, the checkpoints will activate specific signal transduction pathways to stop cell cycle progression until the defects are fixed. It seems to be a paradoxical situation for microbial pathogens facing attack from the host: if the microbial cells must stop cell cycle progression to repair the damage, will they be exposed to the attack for a longer time? Although one would expect that cell cycle checkpoints are likely to be activated when key cellular components of microbial pathogens are being damaged, there has been no report that an impaired cell cycle checkpoint compromises the capacity for infection and virulence of a microbial pathogen. It is also not clear whether drugs targeting the checkpoints of microbial pathogens would significantly lower the chance of their survival in the host. The spindle assembly checkpoint (SAC) has been studied extensively in Saccharomyces cerevisiae. Seven components have been identified essential for its function: MAD1-3 (Li and Murray, 1991), BUB1-3 (Hoyt et al., 1991) and MPS1 (Winey et al., 1991). Disruption of spindle microtubules or the presence of chromosomes unattached to the spindle will activate the checkpoint, which in turn inhibits the anaphase-promoting complex, leading

32 C. Bai et al. to cell cycle arrest (Hoyt, 2000). Cells with a defective SAC will continue to divide even in the absence of spindle, resulting in cell death. Interestingly, deletion of some of the SAC genes, such as MAD2, has no discernible effect on cell growth in unperturbed conditions. This is because chromosome segregation is intrinsically of high precision, and mistakes are rarely made in normal growth conditions. However, pathogens in the host are often exposed to harsh environments that may cause extensive damage to cellular components. Thus, we asked whether the checkpoint functions are required for a microbial pathogen to survive in the host. Candida albicans, a close phylogenetic relative of S. cerevisiae, is the most prevalent fungal pathogen in immunocompromised patients, and systemic infections are often fatal (Odds, 1994). Reports are available that C. albicans yeast cells can grow and switch to hyphal growth within macrophages, which leads to cell death either by rupturing the cell membranes (Lo et al., 1997) or by inducing apoptosis of the macrophages (Schroppel et al., 2001). A mutant defective in the yeast–hypha growth switch was shown to proliferate within macrophages (Lo et al., 1997). These observations strongly indicate the existence of protective mechanisms in C. albicans against killing by host phagocytes. These mechanisms may include both limiting the damage caused by the host and activating the repairing pathways of the pathogen. The success of both mechanisms might require the timely arrest of cell cycle progression. To determine whether the SAC in C. albicans is required for its survival and virulence in mice, we constructed a C. albicans CaMAD2 null mutant. This mutant exhibited a range of phenotypes characteristic of a defective SAC in ways similar to the S. cerevisiae MAD2 mutant. In unperturbed growth conditions, the mutant is indistinguishable from wild-type strains in morphology, doubling time and filamentous growth, except for an increased frequency of chromosome loss. However, the mutant is greatly reduced in viability in both media containing the microtubule toxin nocodazole and peritoneal macrophages of infected mice. It also exhibited significantly reduced virulence in a mouse systemic candidiasis model. Our results demonstrate for the first time that the SAC function is indispensable for C. albicans survival and virulence in the host. Results Functional complementation of S. cerevisiae MAD2 mutant by the C. albicans homologue. MAD2 genes are highly conserved among eukaryotes. The nucleotide sequence of the CaMAD2 gene was retrieved from the C. albicans (strain SC5314) genome

sequence database released by the Stanford Genome Technology Center. In a previous release of the sequence data, there was an open reading frame (ORF) annotated as MAD2. This ORF encodes a putative 213-amino-acid polypeptide. In the latest version of the sequence database, a second ORF homologous to MAD2 is present, which encodes a 215-amino-acid polypeptide. The two ORFs are nearly identical except for a 6 bp insertion in the long version and nucleotide substitutions at two other positions. According to the nucleotide sequences flanking the two MAD2 homologues in their respective cosmids, it appears that this gene is heterozygous in this strain of C. albicans. We aligned the two deduced amino acid sequences with homologues from a diverse range of organisms (Fig. 1). The C. albicans sequences exhibit high homology to all the other sequences throughout the entire alignment, sharing 30–43% identities. The homology is more prominent when the positions containing conserved amino acid substitutions are considered. The long version of the C. albicans homologue has two extra amino acids in a rather variable region. It also differs from the short version at two other positions, one being a conserved substitution and the other a variable position. The short and long copies of the C. albicans MAD2 homologues are designated CaMAD2-1 and CaMAD2-2 respectively. Next, we tested whether CaMAD2 can functionally rescue the defects of the S. cerevisiae MAD2 mutant. The phenotypes of the S. cerevisiae MAD2 mutant have been well characterized, one of which is sensitivity to microtubule-disrupting drugs such as nocodazole (Li and Murray, 1991). The disruption of spindle microtubules normally causes cell cycle arrest before the entry into anaphase, which prevents the disastrous consequence of chromosome mis-segregation (Hoyt et al., 1991; Li and Murray, 1991). In the absence of a functional SAC, such as in the MAD2 mutant, the cell cycle proceeds without a spindle, causing massive mis-segregation of chromosomes and eventually cell death. We cloned both long and short copies of CaMAD2 in a centromeric plasmid driven by the S. cerevisiae MAD2 promoter and transformed them into S. cerevisiae MAD2 mutant cells. Then, the transformants were examined for their sensitivity to nocodazole. Figure 2 summarizes the result. A majority of the wild-type S. cerevisiae cells are arrested with a dumb-bell shape containing a single nuclear mass after 3 h of nocodazole treatment. In contrast, nearly 80% of the MAD2 mutant cells show morphology of three or more connected cell bodies, many of which contain multiple nuclear masses of different sizes, presumably the result of chromosome mis-segregation. Furthermore, ª 90% of the mutant cells lost viability after 4 h of nocodazole treatment; in contrast, few of the wild-type cells © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

Checkpoint function in C. albicans virulence 33

Fig. 1. Alignment of amino acid sequences of Mad2 proteins. The CaMad2p (Ca) sequences were translated from the CaMAD2 nucleotide sequences obtained from the C. albicans genome sequence database (www-sequence.stanford.edu). The complete sequence corresponding to the short CaMAD2-1 allele is shown. Other Mad2p sequences included in the alignment are from: Saccharomyces cerevisiae (Sa; GenBank accession number Z49305), Schizosaccharomyces pombe (Sc; 014417), Arabidopsis thaliana (Ar; NP_189227), Caenorhabditis elegans (Ce; NP_500237), Drosophila melanogaster (Dr; AAF50740), Xenopus laevis (Xe; AAB42527) and Homo sapiens (Ho; NP_002349). The alignment was constructed using the Clustal method in the DNASTAR program. Invariable positions are shaded. Positions conserved in at least five of the eight sequences are denoted by asterisks. The boxes indicated variations in the longer CaMAD2 allele.

© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

34 C. Bai et al. Fig. 2. Functional complementation of S. cerevisiae MAD2 mutant by CaMAD2. A. CaMAD2 rescues the nuclear division and morphological defects of the S. cerevisiae MAD2 mutant caused by nocodazole treatment. Exponential phase cells were treated with 50 mM nocodazole for 3 h in YPD at 30∞C. Nuclei were stained with DAPI. S. cerevisiae strains used are: wild-type A1 (MAD2), MAD2 mutant DRL118.7b (mad), DRL118.7b transformed with S. cerevisiae MAD2 on a CEN plasmid (mad::MAD2), DRL118.7b transformed with either CaMAD21 or CaMAD2-2 on the same CEN plasmid (mad2::CaMAD2) and DRL118.7b transformed with the CEN plasmid alone (mad2::vector). B. Distribution of cells with different numbers of cell bodies after nocodazole treatment for 3 h. C. Percentage viability of cells after nocodazole treatment. Cells were treated as described above, and aliquots were collected at 1 h intervals for the enumeration of the colony-forming units (cfu) on YPD plates.

lost viability after the same treatment. This observation is consistent with the phenotypes reported previously for S. cerevisiae MAD and some other SAC mutants (Hoyt et al., 1991; Li and Murray, 1991). Introduction of either the long or the short copy of CaMAD2 rescued the defects of the S. cerevisiae MAD2 mutant to the same extent and nearly as effectively as the reintroduction of the S. cerevisiae MAD2 gene. This result suggests that CaMAD2 is a homologue of S. cerevisiae MAD2 in both sequence and function. The result also demonstrates that the two CaMAD2 copies are equally functional, consistent with their amino acid differences only in variable positions. Disruption of the CaMAD2 gene We used the URA-blaster method to delete sequentially the two copies of the CaMAD2 gene from C. albicans strain CAI4 (Fonzi and Irwin, 1993). Figure 3A describes the strategy for the gene deletion experiment. Two different gene deletion cassettes, hisG-URA3-hisG (hUh) and cat-URA3-cat (cUc), were used to disrupt the first and second copies of the gene respectively. The correct deletion of the gene after each step was verified by Southern blotting. Figure 3B shows an example of the Southern blotting confirmation of correct gene deletions in the CaMAD2 mutants. From eight heterozygous CaMAD2 mutant clones, we isolated the remaining copy of CaMAD2 by polymerase chain reaction (PCR) for sequence analysis and found that three contained the short (CaMAD2-1/Camad2-2D::hUh) and five contained the long (CaMAD2-2/Camad2-1D::hUh) copy of the gene.

Both types of heterozygous mutants were examined for phenotypes in later experiments. As they exhibited identical phenotypes, they will only be referred to as the CaMAD2 heterozygous mutant below. CaMAD2 mutants are sensitive to nocodazole When grown in GMM or YPD, the Ura+ strains of both heterozygous and homozygous mutants appeared normal, indistinguishable from their isogenic wild-type strains SC5314 and CAF2-1 in morphology and growth rate (not shown). This observation is consistent with the lack of growth defect in S. cerevisiae and Schizosaccharomyces pombe MAD2 and other SAC gene mutants when grown in unperturbed conditions (Hoyt et al., 1991; Li and Murray, 1991; He et al., 1997). To determine whether the CaMAD2 mutants had lost their SAC function, we first tested the sensitivity of the mutants to nocodazole in comparison with CAF2-1. The cells were treated with 50 mM nocodazole (a concentration sufficient to arrest the wild-type cells for about 5 h in YPD medium), and aliquots were harvested after 2 and 4 h to determine cfu on YPD plates. As shown in Fig. 4A, both heterozygous and homozygous mutants were sensitive to nocodazole, with ª 75% of the former and 82% of the latter losing viability after 4 h drug treatment. Reintroduction of either the long or the short version of CaMAD2 into the heterozygous mutants by integration at the RP10 gene locus (see Fig. 5 for a description of the integration experiment) completely restored the resist© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

Checkpoint function in C. albicans virulence 35 Fig. 3. Chromosomal deletion of CaMAD2. A. A schematic description of the strategy for knocking out (KO) the first and second copies of CaMAD2. The first base of the start codon ATG of CaMAD2 is designated as nucleotide (nt) 1. The cleavage sites of PacI and SpeI are shown for the Southern blotting analysis below. B. Southern blotting confirmation of CaMAD2 gene deletion. Two independent clones of each genotype were included. The genomic DNA samples were digested with a mixture of PacI and SpeI. The region corresponding nt –9 to –444 was PCR amplified and labelled by 32P as probe.

ance to nocodazole. However, reintroduction of a single copy of the CaMAD2 gene into the homozygous mutants did not rescue the defect; only when the gene was reintroduced on a multiple copy plasmid (pAB-CaMAD2) were the defects rescued. These results demonstrate that the loss of one copy of the CaMAD2 gene is sufficient to cause an SAC defect in this strain of C. albicans, suggesting that the level of CaMAD2 gene expression is crucial for the function of SAC in C. albicans. A similar observation has recently been reported for the MAD2 gene in mammalian cells: a decrease in the MAD2 expression level resulting from the knock-out of one copy was found to be sufficient to cause the loss of mitotic control (Michel et al., 2001). To confirm that it was not the disruption of microtubules by nocodazole per se but passage through the cell cycle in the presence of the drug that caused the death of CaMAD2 mutants, we first arrested the cells in S phase by treatment with 50 mg ml-1 hydroxyurea for 1 h before the addition of 50 mM nocodazole. Under this condition, the cells remained arrested in S phase (not shown), and the loss of viability was prevented (Fig. 4B). This result shows that the killing of the mutant cells by nocodazole © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

is the continuation of the cell cycle in the absence of functional spindle. To determine the morphology of the nocodazole-treated CaMAD2 mutant cells, the yeast cells treated with the drug for different lengths of time were examined under a microscope. After 2 h nocodazole treatment, nearly all the wild-type cells exhibited a large budded shape with a single nuclear mass, indicating cell cycle arrest (Fig. 6A, a). After 4 h, the wild-type cells grew an elongated bud containing a single nucleus. In a fraction of the cells, the nucleus migrated into the elongated bud. In contrast, after 2 h, both heterozygous and homozygous mutant cells had buds of different sizes (Fig. 6A, b and d), indicating the lack of cell cycle arrest; and after 4 h, most of the mutant cells had three cell bodies or were of irregular shapes. Also, many cells contained multiple nuclear masses of varied sizes. These phenotypes were corrected by the reintroduction of a functional CaMAD2 gene (Fig. 6A, c and e). Again, the phenotypes of the homozygous mutant could only be rescued using the multicopy plasmid. Furthermore, the wild-type cells later exited from cell cycle arrest and grew into branched filaments (Fig. 6B). In contrast, the mutant cells exhibited

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Fig. 4. Sensitivity of CaMAD2 mutants to nocodazole. A. Exponential phase yeast cells in YPD were treated with 50 mM nocodazole at 30∞C, and aliquots were taken after 2 and 4 h to determine cfu on YPD plates. B. The yeast cells were first treated with 50 mM hydroxyurea for 1 h before the addition of 50 mM nocodazole. Strains included are CAF2-1 (filled triangles), Ura+ heterozygous CaMAD2 mutant (open squares), heterozygous CaMAD mutant containing a second copy of CaMAD2 at the RP10 locus (filled squares), Ura+ homozygous CaMAD2 mutant (open circles) and homozygous CaMAD2 mutant transformed with pAB-CaMAD2 (closed circles).

varied degrees of irregular elongation, swelling and branching (Fig. 6C). To determine whether the CaMAD2 mutants are defective in normal filamentous growth, we examined their growth in a number of standard hypha-inducing conditions such as in YPD containing 20% serum and in the mammalian tissue culture medium RPMI at 37∞C. The mutants did not show any discernible defects in hyphal growth (Fig. 6D). When cells arrested in S phase by hydroxyurea were treated with nocodazole for 4 h before being washed and transferred to the hypha-inducing media without the drugs, both wild type and mutants grew into long filaments of similar morphology. This result suggests that the CaMAD2 mutants are normal in hyphal growth in unperturbed conditions, and that the passage through mitosis in the presence of nocodazole caused the abnormal morphology of the mutants. The bud elongation and nuclear migration into the bud during hydroxyurea treatment were also observed by other researchers (Soll et al., 1978; Hazan et al., 2002). Increased frequency of chromosome mis-segregation in CaMAD2 mutants Chromosome mis-segregation occurs at low frequency in normal cells because of the intrinsic accuracy of chromo-

some segregation and the functions of the SAC (Hartwell et al., 1982). Cells with a defective SAC are expected to have an increased frequency of chromosome missegregation even in undisturbed growth conditions. To determine whether the CaMAD2 mutant cells experience an increased frequency of chromosome mis-segregation, we examined the formation of colonies on minimal medium plates containing sorbose as the sole carbon source. Wild-type C. albicans cells cannot grow on such plates, but cells that have lost one copy of chromosome V as a result of chromosome mis-segregation can use sorbose (Sou+) and therefore grow (Rustchenko et al., 1994; Janbon et al., 1998; Magee and Magee, 2000). This provides a convenient assay to determine the effect of a mutation on the frequency of chromosome missegregation. We spread the same number of cells of all strains onto sorbose plates and incubated them at 37∞C for 2 weeks before counting the number of Sou+ colonies. There were ª 20–30 colonies (1 ¥ 106 cells were spread on each plate) on each of the plates containing CAF2-1 and the homozygous mutant rescued by pAB-CaMAD2, whereas hundreds of colonies grew on each of the plates containing the homozygous mutant cells (Fig. 7A). As a control, on glucose plates, all the strains produced similar numbers of colonies. As the Sou+ cells were generated at different times during the whole course of the 2 week incubation, the experiment described above did not measure the rate of loss of chromosome V per cell division; instead, it was used to demonstrate an increased tendency for CaMAD2 null mutants to mis-segregate chromosomes during cell division. Nevertheless, the level of the increase in the number of Sou+ colonies on the mutant plates in comparison with the wild-type plates is largely in agreement with the 15- to 30-fold increase in the rates of chromosome loss in S. cerevisiae MAD mutants (Li and Murray, 1991). The C. albicans mating type locus genes MTLa and MTLa are located on different copies of chromosome V. Thus, the Sou+ cells should contain either MTLa or MTLa alone in the genome (Rustchenko et al., 1994; Janbon et al., 1998; Magee and Magee, 2000). We carried out PCRs using two pairs of oligonucleotide primers, each specific for one MTL gene, to determine the status of chromosome V on 50 Sou+ colonies randomly picked from each plate. For this experiment, we found that it is crucial to streak the Sou+ clones onto sorbose plates a second time to get rid of contaminating live wild-type cells from the first sorbose plate. Figure 7B presents representative results of the PCRs showing the amplified MTL DNA fragments from the Sou+ colonies. All the Sou+ clones derived from either CAF2-1 or the CaMAD2 mutant were found to contain only MTLa or MTLa, confirming the loss of a single copy of chromosome V in the Sou+ clones. Taken together, these results demonstrate a significantly © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

Checkpoint function in C. albicans virulence 37 Fig. 5. Reintroduction of a single copy of CaMAD2 into the CaMAD2 heterozygous mutant by integration at the RP10 gene locus. A. A schematic description of the integration strategy. In the wild-type genomic DNA, CaMAD2 and RP10 genes are located within 12 571 bp and 4121 bp BamHI fragments respectively. A DNA fragment containing the CaMAD2 coding region, its own promoter and 3¢ untranslated region was PCR amplified and cloned between the BamHI and SalI sites of the integration plasmid derived from pCaExp (Care et al., 1999), which contains the RP10 gene for integration and CaURA3 as selection marker. The resultant plasmid, pCaMAD2-int, was linearized by cleavage at the unique StuI site in RP10 and transformed into the Uraheterozygous CaMAD2 mutants. A singlecopy integration at RP10 is expected to cause duplication of the gene with one copy in an ª 8 kb BamHI fragment and another in an ª 3.5 kb BamHI fragment. The former also contains CaMAD2. B. Example of Southern blotting verification of correct integration of a single copy of CaMAD2 at the RP10 locus. The genomic DNA was cleaved with BamHI. The blot was probed with PCR-amplified RP10 and CaMAD2 coding regions separately. Genomic DNA samples from CAI4 (lane 1), a UraCaMAD2 heterozygous mutant (lane 2) and two independent heterozygous clones containing a single-copy integration of CaMAD2 at the RP10 locus (lanes 3 and 4).

increased frequency of chromosome mis-segregation in the C. albicans MAD2 null mutant, consistent with the loss of SAC function. CaMAD2 null mutants exhibit significantly reduced virulence in mice Although CaMAD2 null mutants grow normally in unperturbed conditions, we wanted to know whether they are weakened when exposed to attack by the defence mechanisms of the mammalian host. To examine this, we tested the virulence of the Ura+ homozygous mutant clones using a mouse systemic candidiasis model. The wild-type CAF2-1 and the homozygous mutant rescued by pAB-CaMAD2 were included for comparison. In the group of mice inoculated with 5 ¥ 105 cells, none of the © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

animals injected with the mutant strains died during the 30 day observation, whereas all mice infected with CAF21 or the rescued strains died within 15 days after injection (Fig. 8). When the inoculum was increased to 1 ¥ 106 cells, CAF2-1 and the rescued mutants killed all animals within 8 days, whereas the mutants did not cause death during the 30 day observation. The result demonstrates that, without a functional CaMad2p, the virulence of the pathogen is significantly reduced. Two mice from each of the groups were sacrificed 48 h after infection for histological examination of infected tissues and enumeration of cfu. The kidneys from mice inoculated with 1 ¥ 106 CAF2-1 or the rescued mutant had an average fungal load of 5.24 ± 1.73 ¥ 106 cfu per kidney in comparison with 2 ± 0.46 ¥ 104 cfu per kidney from mice inoculated with the mutant. Microscopic examination of

38 C. Bai et al. Fig. 6. Morphological defects of CaMAD2 mutant cells treated with nocodazole. A. Exponential phase yeast cells were treated with 50 mM nocodazole in YPD at 30∞C and harvested at different times for DAPI staining and microscopy. Cells are shown after 2 and 4 h nocodazole treatment. Strains included are: CAF2-1 (a), Ura+ heterozygous CaMAD2 mutant (b), heterozygous CaMAD2 mutant containing a second copy of CaMAD2 at the RP10 locus (c), Ura+ homozygous CaMAD2 mutant (d) and homozygous CaMAD2 mutant transformed with pAB-CaMAD2 (e). B and C. Photos representative of CAF2-1 and Ura+ homozygous CaMAD2 mutant cells, respectively, after 10 and 13 h nocodazole treatment. D. CaMAD2 mutants show normal hyphal growth in standard hypha-inducing conditions. To induce true hyphal growth, the yeast cells of each strain were treated in RPMI at 37∞C for 4 h. E. Yeast cells were arrested in S phase with 50 mM hydroxyurea in YPD at 30∞C for 1 h before the addition of nocodazole. The cells were incubated further for 4 h before washing and transfer to the hypha-inducing medium RPMI for 4 h. CAF2-1 (a) and Ura+ homozygous mutant (b) are shown.

kidney sections from the animals injected with CAF2-1 or the rescued mutant revealed many areas of fungal growth. In contrast, C. albicans cells were hard to find in corresponding tissue sections from mice inoculated with the mutant (data not shown). Thus, the diminished capacity of the CaMAD2 null mutant for growth and virulence in mice suggests the existence of host conditions that damaged the cellular components or structures monitored by SAC. Growth defects of the CaMAD2 null mutants in mouse inflammatory peritoneal cavity and macrophages The reduced virulence of the CaMAD2 null mutants in mice observed above is probably the result of the loss of cell viability when attacked by host defence. Next, we wanted to examine more closely the behaviour of the CaMAD2 mutant cells when exposed directly to the

inflammatory response in the host. We first injected Freund’s complete adjuvant into the peritoneal cavity of mice to create a localized inflammation. Ten days later, we injected 2 ¥ 108 living C. albicans yeast cells into the enlarged peritoneal cavity and subsequently retrieved aliquots of the peritoneal exudate after 2, 4, 6, 12 and 24 h for microscopic examination of the morphology of C. albicans cells. After 2 h, nearly all the yeast cells from the strains tested were found within macrophages, and some of these engulfed cells have formed short germ tubes (Fig. 9A, a and 9B, a). After 4 and 6 h, longer germ tubes were observed on some cells from both CAF2-1 and the rescued mutants (Fig. 9A, b and B, b). However, most of the germ tubes from the mutant cells were clearly of uneven thickness. By 12 h, the morphological differences between the wild-type and mutant cells became pronounced. Many of the wild-type cells had developed long filaments, obviously having grown out of the © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

Checkpoint function in C. albicans virulence 39 Fig. 7. Increased frequency of chromosome loss in a CaMAD2 mutant. A. The same number of cells of CAF2-1, the Ura+ homozygous mutant and the homozygous mutant rescued by pABCaMAD2 were spread onto minimal medium plates containing either sorbose or glucose as the sole carbon source. Approximately 1 ¥ 106 cells were spread onto each of the sorbose plates and 4 ¥ 102 cells onto the glucose plates. B. PCR confirmation of the loss of chromosome V in the Sou+ clones. The photos show representative results of the PCR amplification of MTLa and a gene fragments from the genomic DNA of the Sou+ clones derived from CAF2-1 (top) and the homozygous mutant (bottom). Only the first nine out of 50 PCRs are shown for clones derived from each strain. CAF2-1 (WT) genomic DNA was included as a positive control.

A

B

macrophages (Fig. 9A, c). In stark contrast, only a small fraction of the mutant cells were in filamentous form, and the filaments were considerably shorter (Fig. 9B, c). Many of the mutant cells were still trapped within macrophages either in the yeast form or with very short germ tubes. By 24 h, the wild-type cells had formed masses of long hyphae (Fig. 9A, d), whereas the mutant cells were hard to find in the exudate at this time point; the few cells that were found looked like residues from cells that had been destroyed by the macrophages (Fig. 9B, d). The cells from the rescued mutants responded like the wild-type strain in the same experiment (not shown). This result demonstrates a remarkable ability of wild-type C. albicans cells to survive attacks by host defence mechanisms in vivo and the dependence of this ability on the presence of a functional CaMAD2 gene. Increased sensitivity of the CaMAD2 null mutant to oxidative stress One important mechanism that macrophages use to © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

destroy the endocytosed pathogens is the release of reactive oxygen species. To test whether the CaMAD2 null mutant is more sensitive to oxidative stress, we tested its sensitivity to H2O2 in culture medium. C. albicans cells were exposed to a range of concentrations of H2O2 in YPD for 1 h before spreading onto H2O2-free YPD for cfu determination. We found that 1% H2O2 killed all wild-type and mutant cells. However, at lower concentrations, the CaMAD2 null mutant exhibited markedly increased sensitivity to H2O2 (Fig. 10). This result suggests that, within macrophages, some of the damage monitored by C. albicans SAC function may also be attributable to reactive oxygen species. Discussion When the cellular components essential for accurate chromosome segregation are damaged, cell cycle progression is normally arrested until the damage is repaired (Hartwell and Weinert, 1989). Cell cycle checkpoints play roles in generating and transducing the signals of damage

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Fig. 8. Reduced virulence of a CaMAD2 null mutant. The virulence of C. albicans strains CAF2-1, the Ura+ homozygous mutant and the homozygous mutant rescued by pAB-CaMAD2 were tested by direct injection of the yeast cells into mice via the tail vein. Fourteen mice were injected for each C. albicans strain at each inoculum, two mice were sacrificed after 2 days for histology, and the rest were monitored for 30 days. Two independent clones of each strain were tested, and the results showed a similar trend.

for the execution of cell cycle arrest. As microbial pathogens are attacked by a range of host defence mechanisms during infection, the checkpoint functions would be expected to be important for pathogens to repair damage that may threaten the integrity of the genome. In this report, we provide experimental results strongly suggesting the importance of intact SAC function for the survival and therefore the virulence of the human fungal pathogen C. albicans. We created C. albicans mutants defective in SAC by deleting the evolutionarily conserved CaMAD2 gene. Like S. cerevisiae and S. pombe MAD2 mutants (Li and Murray, 1991; He et al., 1997), the C. albicans mutants grew normally in unperturbed conditions. However, when treated with the microtubule-disrupting drug nocodazole, which normally activates the SAC, the

mutant cells quickly lost viability. The mutants failed to cause death in mice under the experimental conditions used, indicating greatly reduced virulence of these mutants. The MAD2 gene is among the most extensively studied checkpoint genes in eukaryotes. It apparently has a highly specific function in the cells as an indispensable component of the SAC. This explains why MAD2 mutants from several unicellular organisms exhibit normal growth in unperturbed conditions. Even the Mad2 null blastocysts of mice can grow normally for a considerable time period (Dobles et al., 2000). Although chromosome mis-segregations occur at a markedly increased frequency in the MAD2 mutants, they are still rare events because of the high intrinsic accuracy of chromosome segregation machinery (Hartwell et al., 1982; Li and Murray, 1991). The normal growth of CaMAD2 mutants may also reflect the organism’s higher tolerance of chromosome loss, consistent with the frequent discovery of aneuploid strains (Whelan and Magee, 1981; Chibana et al., 2000). However, serious problems arise when the mutants are exposed to environments that cause damage to spindle microtubules. The cells proceed through mitosis without a functional spindle. This causes extensive chromosome loss and cell death. The reduced ability of CaMAD2 null mutants to grow and cause death in mice is most likely the consequence of cell death because of their failure to arrest cell cycle progression when the cellular events or structures monitored by SAC were disrupted by the host defence mechanisms, a situation similar to the loss of viability of the mutant after nocodazole treatment in culture. The inability of the CaMAD2 null mutants to grow and the loss of viability within the peritoneal macrophages strongly favour this explanation. There are a number of cell cycle checkpoints monitoring the integrity of different cell cycle events and relevant cellular structures. Among them, the DNA damage and replication checkpoints are also very likely to be instrumental for fungal pathogens to survive in the host, because DNA is the primary target of reactive oxygen and nitrogen species, such as hydroxyl free radicals (Nathan and Shiloh, 2000). Failure to arrest cell cycle progression for repair when DNA is damaged will lead to extensive mutations, which can be disastrous to cells. SPK1/RAD53 encodes an essential protein kinase in S. cerevisiae and is required for the activation of both checkpoints (Zheng et al., 1993; Zhou and Elledge, 1993). The C. albicans homologue CaRad53p shares high sequence identity with Rad53p. A recent study identified single amino acid changes in the kinase domain of Rad53p that abolish the checkpoint functions but have no effect on cell growth (Fay et al., 1997). If similar mutations can be constructed in C. albicans, the requirement of the DNA damage and © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

Checkpoint function in C. albicans virulence 41 Fig. 9. Growth defects of a CaMAD2 null mutant in mouse peritoneal exudates and macrophages. Yeast cells from strains CAF2-1 (A) and the Ura+ homozygous mutant (B) were injected into the inflammatory peritoneal cavity of mice. Small amounts of peritoneal exudates were later withdrawn after 2 (a), 4 (b), 12 (c) and 24 h (d) for staining and microscopy. The arrows denote the nuclei of macrophages.

replication checkpoints for the pathogen’s virulence can be tested. With the rapidly increasing cases of C. albicans infections in hospitals, the limited options of antifungal drugs and the widespread occurrence of drug-resistant strains, there is an urgent need to identify novel cellular targets for developing new antifungal therapies. The indispensability of the SAC for C. albicans virulence suggests that the cell cycle checkpoints are worthy of further investigation for this purpose.

Experimental procedures Strains and growth conditions Saccharomyces cerevisiae and C. albicans strains used in our experiments are listed in Table 1. All strains were grown routinely at 30∞C in YPD medium (2% yeast extract, 1%

bactopeptone and 2% glucose). Ura+ transformants were selected on GMM agar plates (2% glucose, 1¥ yeast nitrogen base and 1.5% agar). For looping out the CaURA3 gene within the gene deletion cassette, cells were spread onto a GMM plate supplemented with 1 mg ml-1 5-fluorooratic acid (FOA) and 50 mg ml-1 uridine. Escherichia coli XL1-Blue [supE44 hsdR17 recA1 gyrA46 thi-1 relA1 lac (F¢ proAB lacIqZDM15 Tn10)] was used for cloning experiments and grown routinely on LB agar plates containing 100 mg ml-1 ampicillin.

Functional complementation of S. cerevisiae MAD2 mutants by CaMAD2 The S. cerevisiae MAD2 promoter region from nucleotide (nt) -974 to -1 (the first base of the start codon ATG is nt 1) was PCR amplified from S. cerevisiae genomic DNA (strain A1) with KpnI and BamHI sites added to the 5¢ and 3¢ ends respectively. The CaMAD2 coding sequence (both short and

Fig. 10. Sensitivity of CaMAD2 mutants to oxidative stress. The yeast cells of strains CAF2-1 (a), the Ura+ homozygous mutant (b) and the homozygous mutant rescued by pAB-CaMAD2 (c) were treated with different concentrations of H2O2 in YPD at 30∞C for 1 h before being spread onto H2O2-free YPD plates to determine cfu.


42 C. Bai et al. Table 1. S. cerevisiae and C. albicans strains. Strain

Genotype

Source

S. cerevisiae A1 DRL118.7b ScWY1 ScWY2 ScWY3

MATa MATa MATa MATa MATa

Li and Murray (1991) Li and Murray (1991) This work This work This work

C. albicans SC5314 CAF2-1 CAI4 CaWY1-1 CaWY1-1.1 CaWY1-1.2 CaWY1-1.3 CaWY1-2 CaWY1-2.1 CaWY1-2.2 CaWY1-2.3 CaWY2 CaWY2.1 CaWY2.2 CaWY2.3

Wild type URA3/ura3::l imm434 ura3::l imm434/ura3::l imm434 CaMAD2-1/Camad2-2D::hisG-URA3-hisG CaMAD2-1/Camad2-2D::hisG CaMAD2-1/Camad2-2D::hisG, RP10-CaMAD2-1 CaMAD2-1/Camad2-2D::hisG, RP10-CaMAD2-2 CaMAD2-2/Camad2-1D::hisG-URA3-hisG CaMAD2-2/Camad2-1D::hisG CaMAD2-2/Camad2-1D::hisG, RP10-CaMAD2-1 CaMAD2-2/Camad2-1D::hisG, RP10-CaMAD2-2 Camad2-1D::hisG/Camad2-2D::cat-URA3-cat Camad2-1D::hisG/Camad2-2D::cat Camad2-1D::hisG/Camad2-2D::cat, pAB-CaMAD2-1 Camad2-1D::hisG/Camad2-2D::cat, pAB-CaMAD2-2

his3 leu2 trp1 ura3-52 his leu3 ura3-52 mad2-1 his leu3 ura3-52 mad2-1, CEN MAD2 his leu3 ura3-52 mad2-1, CEN CaMAD2-1 his leu3 ura3-52 mad2-1, CEN CaMAD2-2

long copies) was PCR amplified from C. albicans SC5314 genomic DNA with BamHI and SalI sites added to its 5¢ and 3¢ ends respectively. The promoter fragment was cut with KpnI and BamHI, and the fragment for the coding sequence was cut with BamHI and SalI. These DNA fragments were then ligated into the S. cerevisiae CEN plasmid YcpLac33 (containing URA3 as selection marker) at corresponding sites. The DNA fragment containing the promoter and the coding region of the S. cerevisiae MAD2 gene (nt -974 to the stop codon) was also PCR amplified with BamHI and SalI added to the 5¢ and 3¢ ends, respectively, and cloned in YcpLac33.

Gillum et al. (1984) Fonzi and Irwin (1993) Fonzi and Irwin (1993) This work This work This work This work This work This work This work This work This work This work This work This work

NotI digestion, gel purified and used in the electroporation of CAI4 as described previously (Ramanan and Wang, 2000). Multiple clones of CaMAD2/Camad2D::hisG-URA3-hisG genotype were obtained, and cells were spread onto FOA plates to isolate the Ura- clones of CaMAD2/Camad2D::hisG genotype. To disrupt the second copy of CaMAD2, a new deletion cassette was constructed in which the hisG tandem repeats were replaced by 850 bp E. coli CAT gene repeats. Two independent CaMAD2/Camad2D::hisG clones were transformed with the second cassette to obtain clones of the genotype Camad2D::hisG/Camad2D::cat-URA3-cat. The URA3 gene was then looped out on FOA plates to produce Camad2D::hisG/Camad2D::cat clones.

CaMAD2 deletion The CaMAD2 gene was deleted from strain CAI4 using the URA-blaster method (Fonzi and Irwin, 1993). See Fig. 3A for the construction of CaMAD2 gene deletion cassettes. PCR primers for amplifying CaMAD2 gene segments were designed based on the known CaMAD2 sequence (http://www-sequence.stanford.edu). Primers 5¢-TGCTTCA TCAGAATAATTTTCA-3¢ and 5¢-GAGATCTGGGAAGGGATA GATAGGGTT-3¢ yielded a 435 bp fragment (nt -9 to -444) with a BglII restriction site added to the 3¢ end. This fragment was cloned into pGEM-Teasy (Promega) to generate pMAD2-5¢, in which the BglII site was proximal to the SalI site of the vector. Primers 5¢-GAGATCTTCATTTAGTACAGATATA CA-3¢ and 5¢-GAAGTTTTAGTTAATGAAGCCAT-3¢ amplified a 380 bp fragment (nt 578–958) with the BglII site added to the 5¢ end. This fragment was ligated to pGEM-Teasy to produce pMad2-3¢, in which the BglII site was distal to the SalI site of the vector. The 3¢ segment was then released by BglII and SalI digestion and ligated with BglII–SalI-digested pMad2-5¢. The resultant plasmid was cut open with BglII to accommodate the hisG-CaURA3-hisG fragment with BamHI and BglII ends. The gene deletion cassette was released by

Rescue constructs for CaMAD2 mutants Figure 5 describes the strategy to reintroduce a single copy of the wild-type CaMAD2 gene into the heterozygous CaMAD2/Camad2D::hisG mutant. The coding sequence, together with ª 500 bp of 5¢ and ª 300 bp of 3¢ flanking sequences, was PCR amplified from SC5314 genomic DNA with appropriate restriction sites added to both ends. To reintroduce CaMAD2 into the homozygous CaMAD2 mutant, the PCR fragment was cloned at the BamHI site of the autonomous-replicating plasmid pABSK1 (a gift from H. Chibana, University of Minnesota, USA; see also Ramanan and Wang, 2000) to produce pAB-CaMAD2.

Nocodazole sensitivity The strains to be tested were grown in YPD at 30∞C overnight and then diluted in fresh YPD at a concentration of 5 ¥ 105 cells ml-1 and grown at 30∞C for 2 h before the addition of nocodazole to a final concentration of 50 mM. Aliquots of cells were collected at 1 h intervals, and the numbers of viable © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

Checkpoint function in C. albicans virulence 43 cells were determined by spreading serially diluted samples onto YPD agar plates to score the cfu.

Cell morphology and staining Cells were fixed by the addition of 37% formaldehyde directly to the culture to a final concentration of 3.7% and incubation at 25∞C for 2 h or at 4∞C overnight. The nuclei were stained directly using the Vectorshield mounting medium with DAPI (Vector Laboratories). A Leica DMR fluorescence microscope with Nomarski optics was used, and the images were captured using a Hamamatsu digital camera interfaced with METAMORPH software (Universal Imaging).

Determination of the loss of chromosome V Three 1-day-old-colonies of each strain grown on YPD plates were transferred separately to 500 ml of sterile H2O and washed once. Cell concentration was adjusted to 1 ¥ 106 cells 100 ml-1. One hundred microlitre aliquots were spread onto minimal medium plates containing 2% sorbose as sole carbon source and incubated at 30∞C for up to 14 days to select for Sou+ colonies. To confirm the loss of one copy of chromosome V in the Sou+ cells, two pairs of oligonucleotide primers targeting MTLa and MTLa genes were used for PCR. The pair of primers for detecting MTLa were 5¢-TTGAAGCGTGAGAGG CTAGGAG-3¢ (nt 109–130) and 5¢-ATCAATTCCCTTTCTCT TCGATTAGG-3¢ (nt 595–570); and the primers for MTLa were 5¢-TTCGAGTACATTCTGGTCGCG-3¢ (nt 56–76) and 5¢-TGTAAACATCCTCAATTGTACCCGA-3¢ (nt 571–547). The PCR programme was 95∞C for 5 min followed by 30 cycles of 95∞C for 40 s, 48∞C for 45 s and 72∞C for 1 min.

The mouse model for C. albicans virulence Female Balb/c mice (Jackson Laboratories) aged 8 weeks were used in the virulence test. Cells from each C. albicans strain were grown in YPD to log phase, washed three times and resuspended in PBS at a concentration of 1 ¥ 107 cells ml-1. For each strain, two groups of 14 mice were used, one group was inoculated with 1 million cells per animal and the second group with 2 million cells through the tail vein. Two days after injection, two mice from each group were sacrificed, and kidneys were removed for histology and enumeration of cfu. The rest of the mice were monitored daily for death. One kidney was cut into two coronal pieces, and the other into two sagittal pieces. One piece from each kidney was fixed in 10% formalin for periodic acid–Schiff staining (Sigma), and the other two pieces were homogenized in icecold PBS, and serially diluted samples were spread onto YPD plates for colony counting.

Candida albicans in peritoneal cavity and macrophages of mice Freund’s complete adjuvant (0.4 ml) was injected into the peritoneal cavity of a mouse (Balb/c as described above). After ª 10 days, such mice produced a large amount of © 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 31–44

exudate in the peritoneal cavity as shown by the significantly enlarged abdomen. For each C. albicans strain tested, 2 ¥ 108 yeast cells from late log phase in 200 ml of PBS were injected into the enlarged peritoneal cavity (six mice for each C. albicans strain). Then, 300 ml of the peritoneal exudate was retrieved using a 23G hypodermic needle attached to a 1 ml syringe at 2, 4, 6, 12 and 24 h after the injection. The exudates were smeared onto microscopic glass slides and air dried briefly before fixation in acetone for 10 min. C. albicans cells were stained as follows. The acetone-treated slides were air dried and then immersed in 0.5% periodic acid solution for 5 min before washing in slow running water for 5 min. The slides were covered with Schiff solution (Sigma) and left at room temperature for 15 min. The Schiff solution was poured off, and the slides were washed in running water for 10 min. The nuclei of macrophages were stained by immersing the slides in Harris’s haematoxylin solution (Sigma) for 3 min followed by a 3 min wash in running water. Next, the slides were dipped briefly into a solution containing 70% ethanol and 1% HCl and then washed in running water for 3 min. The samples were dehydrated by immersing the slides sequentially for 5 min each in 70%, 80%, 90% and 100% ethanol followed by 10 min in xylene. The slides were then sealed for microscopic examination.

Determination of C. albicans sensitivity to H2O2 Exponential phase yeast cells (ª 1 ¥ 106 cells ml-1) were treated with a range of concentrations of H2O2 in YPD at 37∞C for 1 h before spreading onto H2O2-free YPD plates to determine cfu.

Acknowledgements We thank W. Fonzi, A. Murray, P. E. Sudbery, A. J. Brown and H. Chibana for generously providing us with C. albicans strains and plasmids, W. J. Hong for critical reading of the manuscript, and members of Y.W.’s laboratory for stimulating discussion. We thank the three anonymous reviewers of the manuscript for their suggestions and comments. Y.W. is an adjunct staff member of the Department of Microbiology, National University of Singapore. The work was supported by the National Science and Technology Board of Singapore.

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