INTRODUCTION

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Candida albicans is a human pathogen of significant medical importance. It can cause life-threatening systemic infections in susceptible individuals, such as those with suppressed immune systems (18). In fact, C. albicans now ranks fourth on the list of most frequently acquired nosocomial bloodstream infections (20).

Hematogenously disseminated C. albicans colonizes diverse organs during infection (18). Several properties have been identified that are likely to be required for successful dissemination and to cause disease, including secretion of degradative enzymes and the ability to switch between the yeast and hyphal growth forms (3, 10, 17). The ability to undergo the yeast-to-hyphal transition appears to be critical for pathogenesis, as mutants unable to form hyphae are less virulent in the hematogenously disseminated mouse model (8, 14, 16). This transition is regulated by signal transduction pathways that respond to environmental signals and stimulate changes in gene expression. One environmental signal that regulates the yeast-to-hyphal transition in vitro is extracellular pH. The ability to respond to extracellular pH appears to be important in vivo. For example, De Bernardis et al. have shown that mutants which are unable to grow at a given pH in vitro are limited in their sites of infection in vivo (5).

We identified two homologs of the RIM101 pathway in C. albicans, RIM101 and RIM8, also referred to as PRR2 and PRR1, respectively (4, 21, 22). In Saccharomyces cerevisiae and Aspergillus nidulans, the zinc finger transcription factor Rim101p (PacCp) is regulated by proteolytic processing in response to environmental pH (15, 19). At alkaline pH, several proteins, including Rim8p (PalFp), stimulate the processing of Rim101p to the active form which governs changes in gene expression (15, 19). In C. albicans, the RIM101 pathway regulates several alkaline responses, including stimulation of alkaline response genes, repression of acidic response genes, and stimulation of the yeast-to-hyphal transition (4, 21, 22). However, the RIM101 pathway is not required for growth at either alkaline or acidic pH in vitro. Because the RIM101 pathway plays an important role in responding to the environment in vitro, we examined the requirement for this pathway in pathogenesis. Here, we show that the RIM101 pathway is required for several host-pathogen interactions and thus pathogenesis. Further, our use of a suppressor mutation has allowed us to narrow the range of RIM101 pathway functions that are required for pathogenesis.

	MATERIALS AND METHODS

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Strains and plasmids. The C. albicans strains used in this study are derivatives of SC5314 and are described in Table 1. Our parent strain, BWP17, is a triply marked auxotrophic strain which we used to generate the rim101/rim101 and rim8/rim8 mutants by PCR directed gene knockout (4, 26). All strains were reverted to prototrophy using plasmid pGEM-HIS1 (26) to create His⁺ strains and plasmid pRS-ARG-URA-BN (see below) to create Arg⁺ and Ura⁺ strains as described below. A prototrophic revertant of our parent strain, BWP17 (26), was generated as follows. First, BWP17 was transformed with NotI-digested pRS-ARG-URA-BN to generate an Arg⁺ Ura⁺ strain. This strain was then transformed with NruI-digested pGEM-HIS1 (26) to generate the prototrophic strain DAY185. DAY44, DAY62, and DAY114 were described previously (4). To generate the prototrophic rim101/rim101 and rim8/rim8 strains, DAY5 and DAY61 (4, 26) were transformed with NruI-digested pGEM-HIS1 to generate the prototrophic strains DAY25 and DAY117, respectively. To generate prototrophic RIM101/rim101 and RIM8/rim8 heterozygous strains, we made DAY2 and BWP35 (4, 26) prototrophic as follows. DAY2 was transformed with NruI-digested pGEM-HIS1 to generate the His⁺ derivative DAY176. DAY176 was then transformed with NotI-digested pRS-ARG-URA-BN to generate the prototrophic strain DAY203. BWP35 was transformed with NotI-digested pRS-ARG-URA-BN to generate the Ura⁺ derivative DAY191. DAY191 was then transformed with NruI-digested pGEM-HIS1 to generate the prototrophic strain DAY205.

rim8/rim8

Media and growth conditions. C. albicans was routinely grown in YPD (2% Bacto Peptone, 1% yeast extract, 2% dextrose). Selection following transformation was done on synthetic medium containing 6.7% yeast nitrogen base with ammonium sulfate and without amino acids, 2% dextrose, and uridine at 80 µg/ml, except when selecting for URA3, and supplemented with the necessary auxotrophic requirements of the cells (1). Cell densities were determined by dilution and counting on a hemacytometer.

Virulence assays. C. albicans strains were passaged overnight at room temperature in YPD at least three times prior to injection. Cells were harvested, washed, counted using a hemacytometer, and resuspended at a density of 2 × 10⁶/ml in sterile phosphate-buffered saline (Irvine Scientific, Irvine, Calif.). BALB/c mice (23 g, male) (B and K Universal, Fremont, Calif.) were infected through the lateral tail vein with 500 µl (10⁶ organisms). Mouse survival was monitored at least twice daily, and moribund mice were euthantized by cervical dislocation. Following injections, dilutions of the fungal suspension were plated on YPD to ensure that similar numbers of viable cells had been injected into the mice. We used C. albicans strain DAY25 to infect 9 mice; DAY44, -62, -106, -203, and -205 to infect 10 mice each; DAY185 and -117 to infect 20 mice each; and DAY114 to infect 21 mice. Statistically significant differences between curves were determined using the Wilcoxon rank sum test. For these analyses, P values of <0.05 were considered to be significant.

Fungal burden. Fungal burdens were determined by injecting 10⁶ C. albicans organisms into the tail veins of 23-g male BALB/c mice (Harlan). C. albicans were prepared and injected as described above. At 20 and 40 h postinfection, the kidneys and livers were removed aseptically from five mice per strain per time point. The organs were weighed, homogenized, diluted in sterile saline, and plated in Sabouraud dextrose agar (Difco). Colonies were counted after incubation of the plates at 37°C for 24 to 48 h, and results were expressed as log CFU per gram of infected organ.

Histopathology. Concomitant with the fungal burden experiment, kidneys were removed aseptically from two mice per strain per time point. Kidneys were fixed for at least 4 h in 100% ethanol at room temperature. They were rinsed and stored in 10% buffered Formalin prior to embedding in paraffin. Thin sections were prepared and stained with hematoxylin and eosin, followed by Gomori methenamine silver, and examined by light microscopy. We found that using both stains allowed visualization of both C. albicans cells and neutrophils within the same section.

Endothelial damage. Candida-stimulated endothelial damage was measured using a chromium release assay described previously (7). The inoculum size was 10⁵ cells/well of a 24-well tissue culture plate. C. albicans and endothelial cells were incubated together for 3 h. Specific release of chromium was calculated using the formula [(2 × experimental release)  (2 × spontaneous release)]/[total incorporation  (2 × spontaneous release)] as described previously (7). All experiments were performed in triplicate and repeated twice.

	RESULTS

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Virulence and the RIM101 pathway. To determine if the RIM101 pathway has a role in virulence, we analyzed the effect of RIM101 pathway mutants in the hematogenously disseminated murine model. Wild-type and mutant strains were injected into mice via the tail vein, and survival of the mice was monitored (Fig. 1A to C). The wild-type and heterozygous RIM101/rim101 strains (DAY185 and DAY203) had similar degrees of virulence, with mice having a 3-day median survival time (Fig. 1A). However, the homozygous rim101/rim101 strain (DAY25) had markedly diminished virulence compared to the wild-type strain, with mice having a 10-day median survival time (P < 0.0001). The complemented rim101/rim101 +RM101 strain (DAY44) was as virulent as the wild type, demonstrating that the virulence defect of the rim101/rim101 mutant is due to loss of Rim101p. Thus, Rim101p is required for virulence in this animal model.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Survival curves of mice following injection with C. albicans. Experiments were done simultaneously but broken up to simplify viewing. Survival was monitored in mice infected with RIM101 mutants (A), RIM8 mutants (B), and RIM8 suppressor strains (C). No mice died between the last time point (day 10) and the end of the experiment (day 14).

rim8/rim8

RIM8/rim8

rim8/rim8

rim8/rim8

rim101/rim101

rim8/rim8 +RIM8

rim8/rim8

rim8/rim8

rim8/rim8 +RIM101-405

rim101/rim101

rim101/rim101 +RIM101

rim8/rim8

rim101/rim101

rim8/rim8

rim101/rim101

rim8/rim8

rim101/rim101 +RIM101

rim101/rim101

rim8/rim8

rim101/rim101

rim8/rim8

Histopathology. Several features of a pathogenic infection are observed in murine kidneys infected with C. albicans (18). To determine whether the RIM101 pathway is required for these responses, we removed kidneys infected with the wild-type strain and RIM101 pathway mutants for histological examination. Forty hours postinfection, three features of a successful infection were seen in kidneys colonized with wild-type and rim101/rim101 +RIM101 cells. First, most wild-type cells had germinated, forming long hyphae within the kidney (Fig. 2B). Second, wild-type cells elicited a strong host immune response, as seen by the number of neutrophils surrounding the C. albicans organisms (Fig. 2A). Third, microabscesses, the sites of concentrated C. albicans and neutrophils, were disseminated throughout the kidney (Fig. 2A). The rim101/rim101 and rim8/rim8 strains showed defects in these assays: there were few organisms, and those that were apparent were often in the yeast form (Fig. 2D and H). Further, there was poor stimulation of the immune response and there were few microabscesses (Fig. 2C and G). Similar results were seen 20 h postinfection (data not shown). We noted that the rim101/rim101 mutant was more defective than the rim8/rim8 mutant by these three criteria (compare panels C and D with G and H in Fig. 2). Thus, both Rim101p and Rim8p are required for host-pathogen interactions leading to normal germination and the development of lesions in the murine kidney.

View larger version (102K): [in this window] [in a new window]   FIG. 2.   Histological samples of kidneys infected with C. albicans for 40 h. Mice were injected with the wild-type (A and B), rim101/rim101 (C and D), rim101/rim101 +RM101 (E and F), rim8/rim8 (G and H), and rim8/rim8 +RIM101-405 (I and J) strains. Sections were visualized using either a 10× (A, C, E, G, and I) or a 40× (B, D, F, H, and J) objective. Arrows denote microabscesses in the 10× panels and representative C. albicans cells in the 40× panels. Note that arrows are absent in panels C and D due to the absence of visible microabscesses and C. albicans cells, respectively.

Endothelial damage. We analyzed the ability of RIM101 pathway mutants to cause damage to endothelial cells in situ. Filler et al. proposed that Candida-stimulated endothelial damage occurs shortly after intravenous infection and is important for entry into the underlying tissues (7). We assayed endothelial cell damage by monitoring ⁵¹Cr release into the medium from lysed endothelial cells in the presence and absence of C. albicans (7). The wild-type, RIM101/rim101, and rim101/rim101 +RM101 strains caused ~35% damage after 3 h of incubation with endothelial cells and were not statistically significantly different from each other (Fig. 3). The rim101/rim101 and rim8/rim8 strains caused 20 and 25% damage to endothelial cells, respectively, a significant reduction compared to the wild type (P < 0.001). Further, the rim101/rim101 strain was more defective than the rim8/rim8 strain (P < 0.05). Endothelial cell damage requires C. albicans germination (9), but we found that all strains, including the rim101/rim101 and rim8/rim8 mutants, germinated equally well (data not shown). Since the RIM101 pathway mutant germinated normally in this assay, these results suggest that the damage defect is due to a downstream event, such as reduced expression of proteases or other factors required for damage (12). Thus, both Rim101p and Rim8p are required to induce wild-type levels of damage to endothelial cells.

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Endothelial damage. Damage to endothelial cells was determined by a chromium release assay. Wild-type and RIM101 pathway mutants were incubated with endothelial cells for 3 h, and release of 51Cr from lysed endothelial cells was measured. Asterisks indicate values that statistically significantly differ from wild-type values (P < 0.001) by analysis of variance.

Suppression of virulence defects. Suppression analysis is a powerful tool with which to identify the cause of a mutant phenotype. The RIM101-405 allele expresses a C-terminally truncated Rim101p which should not require processing for activity and restores filamentation to the rim8/rim8 mutant in vitro (4). We used this allele to elucidate the nature of the rim8/rim8 defect in virulence by genetic suppression studies. First, the rim8/rim8 +RIM101-405 strain was more virulent than the rim8/rim8 strain (P < 0.0001) and was statistically indistinguishable from the wild-type strain or the complemented rim8/rim8 +RIM8 strain (Fig. 1C). Introduction of full-length RIM101 into the rim8/rim8 strain did not have this effect, which demonstrated that this effect is an attribute of the RIM101-405 mutation and not due to increased RIM101 gene dosage. Although the RIM101-405 allele rescued the virulence defect, it did not rescue the fungal burden defect of the rim8/rim8 mutant in kidneys 40 h postinfection (Table 2).

rim8/rim8

rim8/rim8 +RIM101-405

rim8/rim8

rim8/rim8 +RIM101-405

rim8/rim8

rim8/rim8

	DISCUSSION

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Rim101p and Rim8p are required for host-pathogen interactions based upon three assays. First, rim101/rim101 and rim8/rim8 mutants have a severe virulence defect in a murine model. Second, by histological examination, these mutants germinate poorly, fail to stimulate a strong immune response, and fail to form microabscesses disseminated throughout the kidney. Third, these mutants do not stimulate endothelial cell damage to wild-type levels. Thus, the RIM101 pathway is required for the host-pathogen interactions leading to virulence.

Previous in vitro studies of RIM101 pathway mutants did not distinguish the rim101/rim101 and rim8/rim8 mutants (4, 21, 22). Here, we found that although both mutants were defective for all host-pathogen interactions, they do show differences in the three assays. First, the rim8/rim8 mutant is more virulent than the rim101/rim101 mutant. Second, the rim8/rim8 mutant causes more hallmarks of pathology in kidneys than the rim101/rim101 mutant. Third, the rim8/rim8 mutant stimulates more endothelial cell damage than the rim101/rim101 mutant. Thus, we found that both Rim8p and Rim101p are critical for responses in vitro and in vivo but that the assays used in these studies are more sensitive to perturbations of the RIM101 pathway.

Why is the rim101/rim101 mutant less virulent than the rim8/rim8 mutant? Our genetic suppression analyses argue that the function of Rim8p in vivo is to stimulate processing of Rim101p. Studies of both A. nidulans and S. cerevisiae argue that, in the rim8/rim8 mutant, only unprocessed Rim101p is present (15, 19). However, in the rim101/rim101 mutant, no Rim101p is present. Thus, one simple model to explain the difference in virulence is that both unprocessed and processed Rim101p function during infection. The rim8/rim8 mutant has a virulence defect because of the absence of processed Rim101p. The rim101/rim101 mutant has a more severe phenotype due to the absence of both processed and unprocessed Rim101p. We predict that molecular analysis of Rim101p (PacC) pathways in C. albicans and other fungi will support this new hypothesis that unprocessed Rim101p has a functional role in target gene regulation.

In kidneys 20 and 40 h postinfection, the rim8/rim8 and rim8/rim8 +RIM101-405 strains produced similar fungal burdens. However, these strains varied markedly in their virulence. Similar results were reported for other mutants, including the hwp1/hwp1 and chs3/chs3 mutants (2, 13, 25). These results indicate that the fungal burden does not necessarily predict the virulence phenotype. However, these results do suggest that the RIM101 pathway is not required for colonization of the kidney but is required for maintenance of infection.

We did find a strong correlation among the damage, histological, and virulence results. In fact, the histological and damage results appear to predict the virulence result of RIM101 pathway mutants. Similar results are seen for mutations affecting HWP1 and SAP2 (11, 12, 24, 25). Thus, we found that the histological and damage phenotypes are good predictors of the virulence phenotype.

One useful finding to come from these studies is that our wild-type strain appears to maintain normal virulence. We had previously created a triply marked auxotrophic strain to allow for rapid PCR-directed gene knockouts (two markers) and complementation (one marker). Here, we have analyzed host interactions with a prototrophic derivative of BWP17, DAY185. Mice infected with DAY185 appear to have survival times similar to those of mice infected with SC5314 and a related strain, CAI12 (23, 25). DAY185 stimulates endothelial damage to levels similar to those reported for SC5314 (12). Further, DAY185 grows well in kidneys and elicits a strong immune response. Thus, the BWP17 strain is useful to generate mutants rapidly and for consequent in vivo analyses as well.

What is the function of the RIM101 pathway in pathogenesis? Our results argue that the RIM101 pathway is not simply required for growth in vivo. We see that RIM101 pathway mutants have a defect in fungal burden compared to the wild type. However, if we compare the rim8/rim8 and rim8/rim8 +RIM101-405 mutants, which produce similar fungal burdens, we find that the rim8/rim8 +RIM101-405 strain behaves like the wild type in the other assays. Thus, a defect in growth is not sufficient to explain the role of the RIM101 pathway in pathogenesis. The rim8/rim8 +RIM101-405 strain has an activity lacking in the rim8/rim8 strain. We suggest that stimulation of host cell damage may be a candidate for this activity. Filler et al. have suggested that endothelial cell damage may be required to stimulate expression of cytokines and leukocyte adhesion molecules (6). If similar events occur in the kidney, then we predict that mutants that inflict less cell damage would elicit a weaker immune response. This is exactly what we see. Thus, we propose that one function of the RIM101 pathway in pathogenesis is to regulate the expression of genes that stimulate host cell damage.