INTRODUCTION

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Candida albicans is an opportunistic fungal pathogen that causes hematogenously disseminated infections in certain compromised hosts (4, 22). Vascular endothelium may play a critical role during the initiation of hematogenous infection, because blood-borne organisms likely adhere to and penetrate the endothelial cell lining of the blood vessel to gain access to the tissue parenchyma. Furthermore, endothelial cells have the ability to express proinflammatory mediators that may recruit leukocytes to sites of vascular infection (5).

C. albicans can grow in either the yeast or hyphal form, and the ability to germinate and form hyphae is thought to be essential for the virulence of this organism in vivo (2, 19). We have shown previously that germinated organisms interact with host cells, such as vascular endothelium, differently than do blastospores. For example, germinated organisms are significantly more adherent to endothelial cells than are blastospores (23). Also, germination is required for C. albicans to damage endothelial cells and stimulate them to express leukocyte adhesion molecules in vitro (5, 6).

We have been investigating the function of HWP1, a gene that is expressed by hyphae but not blastospores of C. albicans (24). Immunological evidence indicates that this gene encodes a surface protein (26). Deletion of both alleles of HWP1 results in a conditional defect in hyphal formation in vitro (24). Homozygous hwp1 null mutants exhibit almost no germination on all solid media lacking serum. On agar containing serum, the mutants germinate but produce fewer and shorter filaments than do wild-type strains. However, the hwp1 null mutants germinate normally in liquid culture. To evaluate the role of HWP1 in the pathogenicity of C. albicans, we investigated the virulence and morphology of hwp1 null mutants during hematogenous candidal infection in mice. In addition, to investigate potential mechanisms for the diminished virulence of the hwp1 null mutants, we examined the interactions of these organisms with human endothelial cells and neutrophils in vitro.

	MATERIALS AND METHODS

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Organisms. Heterozygous and homozygous hwp1 null mutants were constructed from C. albicans CAI4 by using the hisG URA3 hisG disruption cassette method as described elsewhere (7, 24). Briefly, CAL1 (hwp1::hisG URA3 hisG/HWP1 ura3::imm434/ura3::imm434) was generated from strain CAI4 (ura3::imm434/ura3::imm434). In CAL1, a 434-bp BglII-BamHI fragment in the 5' coding region of one allele of HWP1 replaced with the HisG URA3 HisG disruption construct. The homozygous hwp1 null mutant, CAL3 (hwp1::hisG/hwp1::hisG URA3 hisG ura3::imm434/ura3::imm434) was generated from CAL1 by using the same disruption construct. CAL5, the HWP1 revertant (hwp1::hisG/hwp1::hisG HWP1 URA3 ura3::imm434/ura3::imm434) was constructed from CAL3. The genotypes of all mutants were confirmed by Southern blotting.

Measurement of extracellular proteinase and phospholipase activity. The extracellular proteinase activity of the different strains was measured by using bovine hemoglobin as the substrate by the method of Hube et al. (10). The extracellular phospholipase activity of the various mutants was determined by growing them on egg yolk agar and measuring the size of the zone of precipitation according to our previously described method (13).

Mouse studies. Nine- to eleven-week-old male BALB/c mice (20 to 25 g) were used in this study. They were given food and water ad libitum. Each mouse was inoculated with 10⁶ organisms in 200 µl of PBS via the tail vein. Survival was monitored twice daily, and moribund mice were euthanized by cervical dislocation. To quantify the number of organisms in the tissues, mice were sacrificed after 1, 2, and 3 days of infection by cervical dislocation. Next, 200 µl of blood was obtained by cardiac puncture and plated on Sabouraud dextrose agar. The kidneys and spleen were removed aseptically, weighed, and homogenized in sterile saline, and then serial dilutions were inoculated in Sabouraud dextrose agar. Colonies were counted after incubation at 37°C for 24 h.

Histology. The kidneys and spleens from the mice were fixed in 10% buffered formalin and then embedded in paraffin. Thin sections were prepared and stained with hematoxylin and eosin, as well as Gomori methenamine silver. They were examined by light microscopy. Tissue sections from two mice infected with each mutant were studied.

Endothelial cells. Endothelial cells were harvested from human umbilical veins by the method of Jaffe et al. (14). The cells were grown in M-199 medium supplemented with 10% fetal bovine serum, 10% defined bovine calf serum, and 2 mM L-glutamine with penicillin and streptomycin (5). In all experiments, the cells were grown in multiwell tissue culture plates coated with gelatin and incubated at 37°C in 5% CO₂.

Endothelial cell adherence. The adherence of C. albicans to endothelial cells was determined by our standard method (9). Briefly, 10² blastospores were added to each well of endothelial cells in six-well tissue culture plates and then incubated for 90 min. Virtually all of the organisms germinated during this time. Next, the nonadherent organisms were removed by rinsing in a standardized manner, and the wells were overlaid with Sabouraud dextrose agar. The number of adherent organisms was determined by colony counting. Adherence was expressed as a percentage of the original inoculum, which was confirmed by quantitative culture in Sabouraud dextrose agar. Each experiment was performed in triplicate on three separate occasions.

Endothelial cell phagocytosis of C. albicans. The number of organisms phagocytized by the endothelial cells was measured by a modification of our previously described method (12). Endothelial cells were grown to confluency on 12-mm-diameter glass coverslips coated with human fibronectin. Each coverslip was incubated with 10⁵ organisms for 2 h. Next, the media were aspirated and the cells were fixed with 3% paraformaldehyde in PBS for 30 min. The coverslips were rinsed with 1% bovine serum albumin in PBS (PBS-BSA) and then incubated for 1 h with Texas red-conjugated antiserum directed against C. albicans (Biodesign International, Kennebunk Port, Maine) to label the nonphagocytized organisms. After extensive rinsing in PBS-BSA, the endothelial cell membranes were made permeable by exposing the cells to 0.1% Triton X-100 in PBS. The coverslips were rinsed three times with PBS-BSA and then incubated with 1% Uvitex (a generous gift from Jay Isharani, Novartis, Greensboro, N.C.) in PBS for 30 min (17). This fluorescent chitin stain labeled both the phagocytized and nonphagocytized organisms. Finally, the coverslips were rinsed in PBS-BSA and mounted inverted on microscope slides. The coverslips were examined with a Zeiss Axiovert 10 microscope equipped for epifluorescence. The number of phagocytized organisms was calculated by subtracting the number of nonphagocytized organisms from the total organisms. At least 100 organisms per coverslip were counted, and each experiment was performed in triplicate on three different days.

Endothelial cell damage. The amount of endothelial injury caused by C. albicans was quantified by chromium release assay as described earlier (6). The inoculum was 10⁶ organisms per well of a 24-well tissue culture plate, and the incubation period was 3 h. All experiments were performed in triplicate and repeated three times.

Germ tube elongation. The extent of germ tube elongation by the different mutants on endothelial cells was measured by using a micrometer as described previously (11). Blastospores (5 × 10⁴ organisms per well) in RPMI 1640 medium were added to endothelial cells in 24-well tissue culture plates. After incubation for 3 h, the organisms were fixed in 2% glutaraldehyde, and the lengths of 100 germ tubes of each mutant were measured.

Leukocyte adhesion molecule expression by endothelial cells. Endothelial cells in 96-well plates were infected with 2 × 10⁴ blastospores in RPMI 1640 medium containing 1% fetal bovine serum. Control wells containing the same medium, but no organisms were processed in parallel. After incubating the plates for 8 h, the relative amounts of E-selectin and intracellular adhesion molecule 1 (ICAM-1) expressed by the endothelial cells was determined by whole-cell enzyme-linked immunosorbent assay by the method of Noel et al. (21). The results were corrected for nonspecific binding of the antibodies, which was determined by incubating the wells with the secondary antibody in the absence of the primary antibody. In preliminary experiments, we determined that none of the primary antibodies bound to C. albicans germinated on bare plastic. These experiments were performed in triplicate on three different days.

Neutrophil-mediated growth inhibition of C. albicans. The ability of neutrophils to inhibit the growth of C. albicans on an endothelial cell monolayer was determined by a modification of the method of Meshulam et al. (20). Neutrophils were isolated from heparinized human blood by dextran sedimentation, followed by centrifugation through Ficoll-Hypaque. After removing contaminating erythrocytes by hypotonic lysis, the neutrophils were washed, counted with a hemacytometer, and suspended in RPMI 1640 medium containing 10% pooled human serum (Sigma Chemical Co., St. Louis, Mo.). These leukocytes were added to endothelial cells in a 48-well tissue culture plate that had been infected for 3 h with 2.5 × 10⁴ C. albicans blastospores per well. The ratio of organisms to neutrophils was 1 to 4. After a 1-h incubation, the neutrophils and endothelial cells were lysed with distilled water, and the number of metabolically active organisms was determined spectrophotometrically by the reduction of the water-soluble tetrazolium salt, 2,3-bis(2-methoxy-4-nitro-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (20). The number of organisms inhibited by the neutrophils was calculated from a standard curve, which was constructed by incubating known numbers of organisms with endothelial cells in the absence of neutrophils. These experiments were repeated twice in triplicate.

Data analysis. Differences in survival of the mice infected with the various strains were analyzed using the Wilcoxon rank sum test. In all other experiments, differences were compared using analysis of variance. When multiple comparisons were performed, the Bonferroni correction was used. P values of 0.05 were considered significant.

	RESULTS

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Virulence of C. albicans strains in mice. The hwp1 null mutants exhibited defects in hyphal formation in vitro (24). Hyphal formation has been implicated in the virulence of C. albicans (19). Therefore, the contribution of HWP1 to the virulence of C. albicans in the murine model of hematogenously disseminated candidiasis was determined. Mice infected with the control strain, CAI12, survived a median of 3.5 days (Fig. 1). CAL1, the heterozygous hwp1 null mutant, was significantly less virulent than was CAI12, since mice infected with CAL1 had a median survival of 10 days (P < 0.001 compared to CAI12). The virulence of the homozygous hwp1 null mutant, CAL3, was even less than that of CAL1. The median survival of mice infected with CAL3 was greater than 14 days (P < 0.001 compared to CAI12 and CAL1). Similar results were obtained with a second homozygous hwp1 null mutant that was constructed independently of CAL3 (data not shown). These results suggested that the HWP1 gene product is important for the virulence of C. albicans. Also, the relationship between the number of functional alleles of HWP1 and in vivo virulence suggested a gene dosage effect in this animal model of infection.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Survival of mice following intravenous challenge with 106 blastospores of CAI12 (n = 10), CAL1 (n = 20), CAL3 (n = 33), and CAL5 (n = 24).

Quantitative culture results. All strains tested produced a similar level of infection in the kidneys after 1 day of infection (Fig. 2). However, after 2 and 3 days of infection, the kidneys of mice infected with either the heterozygous or the homozygous hwp1 null mutants (CAL1 and CAL3, respectively) contained significantly fewer organisms than did those of mice infected with either CAI12 or the HWP1 revertant, CAL5. In addition, after 3 days of infection, there were significantly fewer organisms in the kidneys of mice infected with CAL3 compared with those infected with CAL1. Finally, the number of organisms in the kidneys of mice infected with CAL3 decreased progressively after the first day of infection. In contrast, the number of organisms in the kidneys of the mice infected with the other strains either remained constant or increased during the course of the study.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Colony counts of the kidneys of mice infected with different strains of C. albicans. Seven mice were infected with each strain. Results are the mean ± the standard deviation. *, P  0.001 compared to CAI12; , P < 0.001 compared to CAL3.

Histopathologic examination of the kidneys. Because deletion of one or both copies of HWP1 reduces the ability of C. albicans to germinate on solid media in vitro (24), we investigated the germination of the different hwp1 mutants in vivo after 48 h of infection. The majority of the organisms were in the hyphal phase in the kidneys of the mice infected with the hwp1 heterozygote, CAL1 (Fig. 3). In the kidneys of the mice infected with the hwp1 homozygote, CAL3, the fungi appeared to be a mixture of ovoid and elongated cells. The longest hyphae were observed in the kidneys of the mice infected with the HWP1 revertant, CAL5. Thus, the extent of hyphal formation of HWP1 mutants in the kidneys of mice paralleled their virulence.

View larger version (129K): [in this window] [in a new window]   FIG. 3.   Micrographs of organisms in the kidneys of mice at 48 h postinfection. Mice were infected with 106 blastospores of CAL1 (a), CAL3 (b), and CAL5 (c). Sections of the kidney were stained with silver (magnification, ×224). Arrows indicate C. albicans.

Interactions of the hwp1 mutants with endothelial cells and neutrophils in vitro. To investigate potential mechanisms for the diminished virulence of the hwp1 mutants, we examined the interactions of the different strains with endothelial cells and neutrophils in vitro. We found that deleting both copies of hwp1 had a significant effect on only a few of these interactions. The homozygous hwp1 null mutant, CAL3, produced shorter germ tubes and caused somewhat less endothelial cell injury than did either CAL1 or CAL5 (Table 1). Although CAL3 produced shorter hyphae on endothelial cells than did the other strains, the percentage of the homozygous hwp1 mutants that germinated on these cells was similar to that of the other strains (data not shown). In addition, CAL3 was phagocytized by endothelial cells only slightly less efficiently than were the two other strains. Furthermore, although lytic enzymes released by the fungi likely contribute to endothelial cell injury (12), the extracellular proteinase and phospholipase activities of all strains studied were similar (data not shown).

	DISCUSSION

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In C. albicans, the formation of hyphae or pseudohyphae is triggered by a diverse set of environmental signals, and different genes appear to regulate this morphologic transformation. The majority of investigations on the regulation of filamentation in C. albicans have focused on genes that regulate this process in vitro. However, it is possible that different genes are important in regulating filamentation in vivo. Therefore, investigating the role of HWP1 in candidal germination in the murine model of hematogenous infection complemented our in vitro studies. We found that hwp1 mutants exhibited marked defects in germination in the murine kidney following intravenous infection. This reduction in germination was accompanied by a significant decrease in virulence. The role of the HWP1 gene product in the formation of filaments in vivo is consistent with our findings that hwp1 null mutants are defective in filamentation on solid media in vitro (24).

When tested in vitro, the hwp1 mutants germinated normally in liquid media containing 10% serum but exhibited reduced hyphal development when grown on serum-containing agar (24). In the murine kidney, the organisms were almost certainly exposed to serum constituents. The finding that the null mutants germinated very poorly in vivo suggests that the signal transduction pathway(s) that induces germination on solid media may also regulate germination in the murine kidney.

To date, at least two distinct signal transduction pathways that induce the yeast to hyphal transformation in C. albicans have been identified. One pathway contains the transcription factor encoded by EFG1, and the other is the mitogen-activated protein (MAP) kinase pathway (1, 15, 16, 18, 19, 27). HWP1 expression is dependent on EFG1 in vitro, because there is no detectable HWP1 expression in a homozygous efg1 null mutant (24). In vivo, HWP1 may be regulated by factors other than Efg1p because the hwp1 mutants had markedly reduced virulence, whereas the virulence of efg1 mutants appears to be diminished to a lesser extent (19). However, because the virulence of the different mutants of C. albicans was assessed in different strains of mice, this conclusion requires additional experimental confirmation.

HWP1 also appears to be regulated independently of the MAP kinase pathway in vitro and in vivo. We have found that, in vitro, HWP1 is expressed at normal levels in a homozygous cph1 mutant, which lacks the terminal transcription factor of the MAP kinase pathway (24). In the murine model, the hwp1 null mutants also had a different phenotype from any of the MAP kinase mutants. The hwp1 mutants exhibited deficient germination in the murine kidney. In contrast, although some of the MAP kinase mutants have reduced virulence in vivo (1), in the one study where histopathology was performed, no reduction in filamentation was seen in the tissues (16). Thus, the reduction in virulence of the MAP kinase mutants may be caused by factor(s) other than decreased filamentation.

The C. albicans gene, INT1, resembles HWP1 in that its product appears to be a surface protein that plays a role in hyphal formation (8). In vitro, int1 null mutants germinate normally in liquid media but have reduced germination on solid media. Like the hwp1 mutants, int1 mutants are less virulent in the murine model of hematogenously disseminated candidiasis. However, it is unknown whether the reduced virulence of the int1 mutants is due to impaired hyphal formation because the ability of these mutants to germinate in vivo has not been reported. Furthermore, the signal transduction pathways that regulate int1 expression are currently unknown.

When testing the virulence of the hwp1 mutants, we saw evidence of a gene dosage effect. The virulence of the heterozygous mutant, CAL1 was intermediate to that of the wild-type strain and the homozygous null mutant, CAL3. This gene dosage effect was also evident in the ability of the organisms to form hyphae in vivo and in vitro (24).

An unexpected finding was that the HWP1 revertant, CAL5, was as virulent as the parent strain. Phenotypic differences between CAL1 and CAL5 were also observed in our in vitro studies (Table 1) (24). These phenotypic differences were not likely due to extraneous mutations because the differences were reproducible in independently constructed revertants. The results of the Southern blotting precluded the possibility that any of the revertants contained more than one copy of HWP1 (24). However, it is possible that the fragment of HWP1 that was used to construct the revertant did not contain the full-length promoter, and the presence of this incomplete promoter resulted in higher-level expression of the reintroduced allele. Alternatively, the structure of the reverted locus in CAL5 may have been altered in such a way that the expression of HWP1 was increased. Nevertheless, the finding that reintroduction of HWP1 into the homozygous null mutant reversed its filamentation and virulence defects demonstrates that the deletion of HWP1 was directly responsible for these defects.

When investigating the number of organisms in the kidneys, we found that significant differences among the various hwp1 mutants were observed only after 2 days of infection but not at the earlier time point (Fig. 2). These results may indicate that germination or another process associated the HWP1 gene product is required for the organism to persist in the kidney and avoid clearance by host defense mechanisms. They also suggest that initiation of a renal infection can occur independently of the HWP1 gene product.

Recently, Staab et al. (25) reported that the Hwp1 protein may act as an adhesin by serving as a substrate for host cell transglutaminases. As with our findings, these authors determined that hwp1 null mutants of C. albicans are less lethal in the murine model of intravenous infection. Thus, it is possible that the reduction in virulence of the hwp1 null mutants may be due to a decrease in the ability of these mutants to adhere to host cells, as well as a reduction in their capacity to form hyphae.

Although the different mutants produced significantly different organism burdens in the kidneys, all mutants tested achieved similar levels of infection in the spleen and blood. This finding suggests that candidal factors other than the HWP1 gene product are required for the maintenance of infection in these anatomic sites. Evidence of niche-specific virulence factors of C. albicans has been reported previously with the PHR1 and PHR2 gene products (3).

To develop in vitro correlates of the in vivo pathogenicity of the different mutants, we examined their interactions with endothelial cells and neutrophils. These in vitro studies demonstrated some differences among CAL1, CAL3, and CAL5 (Table 1). For example, CAL3 produced the shortest germ tubes and caused the least amount of endothelial cell injury. We have found previously that a sap2 mutant of C. albicans that is deficient in secreted aspartyl proteinase 2 causes less injury to endothelial cells in vitro (12). This mutant also has attenuated virulence in two different animal models (10). Therefore, it is possible that endothelial cell injury in vitro may be useful as a surrogate marker for virulence in animal models of hematogenously disseminated infection.

We also observed that when the mutants were grown on endothelial cells, the average germ tube length of CAL5 was significantly shorter than that of CAL1. This result is the opposite of what was observed in vivo. The germ tube elongation experiments were performed in RPMI 1640 medium. We repeated them in M199 medium containing 20% bovine serum to determine if a richer medium might give results more similar to those obtained in vivo. However, even in this medium, CAL1 still produced longer germ tubes than did CAL5 (data not shown). These findings indicate that the in vitro conditions do not always elicit the same candidal responses as were induced in the murine model. This difference between the in vitro and in vivo responses may also explain why there was no detectable difference in the susceptibility of the different mutants to neutrophil-mediated inhibition in vitro, even though the homozygous hwp1 null mutant appeared to be cleared more rapidly from the kidneys in vivo.

In conclusion, our results indicate that the HWP1 gene product is important for both the germination and pathogenicity of C. albicans in vivo. These findings provide strong evidence for the importance of filamentation in candidal virulence and suggest that the yeast-to-hypha transition may enable the fungus to avoid host clearance mechanisms in some organs. Therapeutic strategies that inhibit this morphological change may be useful adjuncts for treating serious infections caused by C. albicans.