Profile of Candida albicans-Secreted Aspartic Proteinase Elicited during Vaginal Infection
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感染与免疫杂志 2005年第3期
Institute of Clinical Microbiology, Immunology, and Hygiene, Friedrich-Alexander Universitt Erlangen-Nürnberg, Erlangen
Institut für Molekulare Infektionsbiologie, Universitt Würzburg, Würzburg, Germany
ABSTRACT
Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.
INTRODUCTION
Candida albicans is a common commensal fungal organism that is part of the normal microflora of the genital and gastrointestinal tract of healthy individuals. Under appropriate conditions, C. albicans can become an opportunistic pathogen that has debilitating and sometimes deadly consequences for its host (8). One common manifestation of Candida infection is superficial candidiasis, which is a significant problem encountered in clinics worldwide. The most common superficial infections are vaginal candidiasis and mucosal candidiasis of the oropharyngeal cavity and the esophagus (reviewed in reference 3).
Vulvovaginal candidiasis (VVC) affects an estimated three of four women at least once during their reproductive years, and by the age of 25 one-half of all college women will have had at least one physician-diagnosed episode (12, 13, 22, 39). Although some women never contract VVC and others experience only sporadic acute episodes, a third significant subpopulation comprising as many as 5% of all adult women experience subsequent recurrent VVC (10, 31). Although several exogenous factors are thought to increase the incidence of VVC (pregnancy, oral contraceptives, diabetes mellitus, and antibiotics) (38, 40), the pathogenesis of recurrent VVC is still largely unknown.
Several factors of C. albicans were previously identified as virulence determinants; these factors include hyphal formation (41), phenotypic switching (17, 43, 49), and secretion of hydrolytic enzymes (14, 19, 32). Although hyphal formation is probably the best-documented virulence determinant of C. albicans, the production of hydrolytic enzymes, specifically the secreted aspartic proteinases (Saps), as key virulence determinants has been comprehensively studied (4). Ten SAP isogenes encode the Sap proteins (7, 26, 27). The purported functions of Saps during infection include the digestion of host proteins for nutrient supply, evasion of host defenses by degradation of immunoglobulins and complement proteins, adherence, and degradation of host barriers during invasion (19, 20, 23, 35, 44, 50). Due to their wide substrate specificity and broad pH range (2), it is accepted that Saps contribute to the development of active Candida infections.
Previous studies have highlighted the importance of certain SAP genes in relation to various models of infection (21, 29). Furthermore, individual members of the gene family might have their own unique roles in the infectious process because environmental conditions have been shown to affect production of Saps differentially, resulting in a niche-specific expression pattern of SAP genes (14, 16, 18-20, 23, 35, 44, 48, 50). An in vivo expression technology (IVET) that is based on the irreversible deletion of a mycophenolic acid resistance (MPAR) marker from the genome as a stable reporter of gene activation has proven to be very useful for detecting, on a single-cell basis, the induction of genes that are only transiently expressed at a certain stage of infection. By using this system, differential activation of individual members of the SAP gene family was observed in esophageal infections, in intraperitoneal infections, in gastrointestinal infections, and after hematogenous dissemination to the kidneys (23, 44).
Studies investigating the production of SAP mRNA and the virulence of SAP knockout strains during Candida vaginitis have been performed previously. By using an in vitro reconstituted human vaginal epithelium model, detection of SAP1, SAP4, and SAP5 was found to coincide with the onset of significant tissue damage (34). Additional studies have documented the expression of SAP1, SAP3, and SAP6 to SAP8 during human vaginal infection by using reverse transcription (RT)-PCR. In a rat model of Candida vaginitis, the importance of SAP1 to SAP3 was demonstrated by using specific knockout strains of C. albicans (5, 30).
In this study, we examined the SAP expression profile that is exhibited by C. albicans in an experimental mouse model of vaginal candidiasis and how this expression profile correlates with hyphal formation. We used two different genetic reporter systems to detect the activation of individual SAP promoters by host signals, the recombination-based IVET system and C. albicans strains expressing the GFP reporter gene under control of the promoters of selected SAP genes. This allowed us to microanalyze the temporal and spatial expression pattern of SAP genes in single hyphal segments and to test our hypothesis that a specific SAP expression profile is also elicited in this host niche. To our knowledge, this is the first study in which reporter strains were used to analyze both SAP induction and its relationship to hyphal formation in an in vivo model of vaginal candidiasis.
MATERIALS AND METHODS
Strains and growth conditions. All strains were maintained on complete supplement medium without uridine (CSM-ura) agar plates containing (per liter) 6.7 g of yeast nitrogen base without amino acids (Bio 101), 2 g of glucose, and 0.77 g of CSM-ura (Bio 101). For in vivo studies, C. albicans strains were grown to the stationary phase in CSM-ura liquid medium for 16 to 18 h at 28°C on an orbital shaker. After two washes with sterile water, the number of blastoconidia was determined by using a hemocytometer, and blastoconidia were transferred with pipettes directly into the vaginal canal.
The C. albicans strains used in this study and, for clarity, the relevant parent strains are described in Table 1. The C. albicans IVET system has been described previously (44, 47). Briefly, the reporter strains contain the ecaFLP gene, which encodes the site-specific recombinase FLP, under the control of one of the SAP promoters at the original genomic locus and a genomically integrated MPAR marker that is flanked by direct repeats of the FLP recognition sequence (FRT). Induction of the SAP promoter results in excision of the MPAR marker from the genome, so that the corresponding cell and its progeny grow more slowly and form smaller colonies than their parent on agar plates containing a suitable concentration of mycophenolic acid (MPA) (1 μg/ml). For each of the SAP genes (SAP1 to SAP6), two independently generated reporter strains were used which carried the reporter fusion in one of the two possible alleles (e.g., SAP2-1 and SAP2-2) whenever the alleles could be distinguished by restriction site polymorphism and by sequencing of the cloned promoters (Table 1). As controls for the MPA-resistant and -sensitive phenotypes, a strain containing a stably integrated MPAR marker (S2UI1A) and a strain that did not contain the MPAR marker (S2FI1AS) were used. In all in vivo experiments, appropriate dilutions of the inocula were plated on MPA indicator plates to determine the proportion of MPA-sensitive cells before infection, which was always less than 3%. To detect SAP activation during infection, vaginal lavage fluids obtained at different times after inoculation were spread on MPA indicator plates. After incubation at 30°C for 3 to 4 days, the percentage of small colonies was determined. Because the maximum proportion of small colonies in the inocula was 3%, we defined an arbitrary limit of 9% MPA-sensitive colonies (three times the nonspecific background proportion) as a threshold for significant induction of a specific gene.
In addition, a series of strains which carried the GFP gene as a reporter was generated (Morschhuser, unpublished data). Briefly, a C. albicans-adapted GFP reporter gene (28) was substituted for the ecaFLP gene in the corresponding plasmids. In addition, a similar construct was made in which the GFP gene was placed under control of the strong ADH1 (alcohol dehydrogenase) promoter. The reporter fusions were then integrated into the respective loci of parental strain CAI4 (Table 1). Inocula of GFP-expressing strains were microscopically examined to verify that GFP expression had occurred only in cultures of the control strain harboring the ADH1 promoter-GFP fusion and that no detectable GFP expression had occurred in the cultures with SAP promoter-GFP fusions before infection. Validation of green fluorescent protein (GFP) as a reporter of gene expression in vivo has been extensively demonstrated recently (1).
In vivo infection studies and recovery of vaginal C. albicans. Eight- to ten-week-old female BALB/c mice were obtained from Charles Rivers Breeding Laboratories (Sulzfeld, Germany). To induce pseudoestrus during infection, mice were injected subcutaneously with 0.02 mg of estradiol valerate (Sigma Chemical Co., St. Louis, Mo.) per ml in 0.1 ml of sesame oil 72 h before inoculation with C. albicans (9, 33, 42). Estradiol treatments were continued at weekly intervals thereafter. Infection of the vaginal canal was initiated by inoculating mice intravaginally with 5 x 104 stationary-phase cells of a strain in 20 μl of phosphate-buffered saline (PBS) (9). Mice were either sacrificed or anesthetized, and vaginal lavages were performed at different times following infection. We included controls which showed that the use of live or sacrificed mice for vaginal lavage did not affect the number of CFU obtained or the outcome of the infection.
For experiments involving FLP reporter strains, mice were anesthetized and lavaged with 50 μl of sterile PBS on days 1, 4, 9, 14, 20, 27, and 32. Lavage fluids were serially diluted and plated, and the percentage of MPA-sensitive cells was determined (total number of MPA-sensitive cells/total number of CFU x 100). Experiments were repeated at least twice with independently generated reporter strains for each SAP gene.
Studies comparing the induction of SAP5 with the number of CFU, hyphal formation, and hyphal length were performed as follows. Mice were sacrificed 1, 2, 4, 6, 8, 12, 23, 36, and 50 h postinfection and lavaged with 100 μl of sterile PBS. Fifty microliters of lavage fluid was serially diluted and plated, and the total number of CFU and the percentage of MPA-sensitive cells were determined. The remaining vaginal lavage fluid was subjected to microscopic examination as described below.
For experiments involving GFP reporter strains, mice were anesthetized and lavaged with 50 μl of sterile PBS on days 1, 4, 9, 14, 21, and 28. Immediately following each lavage, samples were subjected to microscopic examination as described below. Experiments were repeated with two independently generated reporter strains for each SAP gene.
Microscopic examination. For microscopic examination of the cellular morphology of C. albicans FLP reporter strains during infection, vaginal lavage samples from sacrificed mice were solubilized by using a 20% KOH solution at room temperature. This step was included because of the viscosity of the samples due to the presence of large amounts of mucus in the vagina. Specimens were centrifuged (1,500 x g for 10 min), and the sediment was mounted on glass slides in the presence of fluorescent brighteners (Calcofluor white; Sigma) for epifluorescence microscopy (Zeiss Axiophot). Approximately 100 cells from multiple slides were observed to determine the percentages of hyphae and blastospores in the vaginal lavage fluid. Images of representative fields were acquired with a SPOT camera, and measurements were taken with the MetaView software package to determine the total hyphal length and the length of the individual hyphal segments.
GFP expression was examined directly following vaginal lavage of anesthetized mice. Thin wet-mount slides (each containing 5 μl of the vaginal lavage fluid) were prepared. Between 100 and 200 individual cells, depending on the vaginal fungal burden, were evaluated for GFP expression by epifluorescence microscopy. Representative images for conventional phase-contrast microscopy and consecutive fluorescence exposures of identical fields were acquired with the MetaView software package. Overlays created by merging the two images are presented below.
Statistical analysis. The unpaired Student t test was used to analyze the data when applicable. Significant differences were defined as a confidence level at which the P value was <0.05. Unless indicated otherwise, data are expressed as means ± standard errors of the means. Correlations between increases in average hyphal length either for time after infection or for increases in the average percentage of MPA-sensitive cells were analyzed by using linear regression by minimizing the sum of squares.
RESULTS
Analysis of SAP expression in vaginal candidiasis. The recombination-based IVET is very useful for measuring cumulative gene expression because cells that have lost the MPAR marker by FLP-mediated recombination after induction of the target gene promoter remain MPA sensitive even when the promoter is down-regulated again at later stages of the infection. We determined the in vivo SAP expression profile of C. albicans that is elicited during a vaginal infection on a single-CFU basis. After induction of pseudoestrus, the mice were infected with SAP1 to SAP6 reporter strains and two controls (see Materials and Methods). Mice were lavaged on days 1, 4, 9, 14, 20, 27, and 32, and the lavage fluids were plated to determine the percentage of MPA-sensitive colonies.
Figure 1 shows the results of two experiments for three representative days of the 32-day experiment (days 4, 14, and 27). By day 4 postinfection, the SAP5 promoter had been detectably activated in approximately 100% of the cells recovered from the vagina. In fact, SAP5 induction was observed in 48% ± 10% of the cells as early as day 1 after infection. SAP4 activation was also detected early in the infection, and there were only slight differences in the two clones tested. By day 4 postinfection, we detected induction of SAP4 in 25% ± 10% of the C. albicans cells recovered from vaginal lavage fluids. By day 14 postinfection, all mice infected with the SAP4 or SAP5 reporter strains contained significant proportions of MPA-sensitive cells (Fig. 1B), and by day 27 after infection 100% of the colonies from these mice were MPA sensitive (Fig. 1C). Surprisingly, in contrast to findings of other studies in which vaginal infections were evaluated, no other SAP genes examined were highly or consistently induced at detectable levels over the course of the 32-day vaginal infection.
SAP5 expression occurs after a matter of hours and correlates with hyphal formation. Since we observed such high and early activation of SAP5, we investigated the onset of induction by examining earlier times during infection, starting 1 h postinoculation. Lavage fluids were plated, and a portion of each preparation was used for microscopic examination of morphogenic development. In this way, we assessed if SAP5 expression correlated with the fungal load and/or hyphal formation in the vagina.
To address the first point, we determined the average number of CFU obtained from three mice per time and plotted the average number of CFU against the rise in the number of MPA-sensitive colonies. As illustrated in Fig. 2A, there was an initial drop in the number of CFU following inoculation, but thereafter the total number of CFU grown from the vaginal lavage fluid remained relatively constant throughout the 50 h of observation (between 2.3 x 103 ± 1.4 x 103 and 1.1 x 104 ± 0.6 x 104 CFU). However, the percentage of MPA-sensitive colonies increased in some mice (Fig. 2B) to 17% as early as 2 h after infection, and the mean level of SAP5 induction rose sharply after 12 h (significance for the group of animals reached between 24 and 36 h, P < 0.03).
With regard to the second point, we examined the correlation between hyphal formation and SAP5 induction by comparing the proportion of MPA-sensitive colonies with the morphology of the cells in the samples. As shown in Fig. 2B, expression of SAP5 was variable. In some animals, it was detectable after 2 h of infection, and expression was reproducibly detected between 12 and 23 h. After examination of the lavage fluids for the presence of hyphae (Fig. 2C), we found that substantial SAP5 activation was detected only in lavage samples in which the majority of cells were in the hyphal form, but it must be emphasized that not all samples with significant amounts of hyphal cells gave rise to MPA-sensitive colonies, because SAP5 expression was not observed in every sample in which hyphae predominated over yeast cells. For example, after 2 h of infection the mouse 1 (M1) sample contained 78% hyphae, and 17% of the colonies were MPA sensitive, well above the background values. In contrast, strong hyphal induction (71% hyphae) was also observed in the M2 sample after 4 h of infection, but only 3% MPA-sensitive colonies were detected in this sample, a value which is similar to the background values in the inocula before infection. None of the samples containing a majority of blastospores (M1 sample at 1 h; M2 and M3 samples at 2 h; M3 sample at 4 h; M1 sample at 6 and 12 h) gave rise to a considerable number of MPA-sensitive colonies, indicating that the level of SAP5 induction was below the detection limit of the reporter system in these samples.
SAP5 induction correlated with the presence of hyphae but not with hyphal length. The mean hyphal length increased constantly over time, indicating that there was a constant rate of growth and propulsion of filaments (r2 = 0.91, P 0.03). However, the increase in the number of MPA-sensitive cells did not correlate with hyphal length (r2 = 0.19, P > 0.05).
Detection of SAP expression during vaginal infection by using the GFP reporter gene. In order to confirm the results obtained with the FLP reporter strains, we tested if GFP could be used as an alternative reporter of gene expression in the mouse model of Candida vaginitis. Preliminary experiments were performed with strains that constitutively expressed GFP from the ADH1 promoter. As shown in Fig. 3A, even in the presence of cellular infiltration and large amounts of mucus distinct hyphal cells were distinguishable from background fluorescence, and a detailed hyphal structure could be observed. These results suggested that GFP is a suitable reporter for analysis of gene expression in this infection model and that it could even be used to detect possible differences in SAP induction in individual hyphal compartments of the same filament.
We then investigated the expression of SAP4 and SAP5 during vaginal infection using C. albicans strains that carried chromosomally integrated SAP-GFP fusions (see Table 1 for strain descriptions). Since we were unable to detect expression of the SAP2 gene in this infection model with the recombination-based IVET, we also included two reporter strains in which GFP was expressed from the promoters of either of the two SAP2 alleles, which have been shown to differ in their inducibility in this strain background due to allelic differences (45).
As shown in Fig. 3B, strong SAP5 promoter activity was observed as early as day 1 and persisted through day 28 following infection. SAP4 expression was detected starting on day 4 postinfection and was present through day 28. In contrast, and in agreement with the results obtained with the FLP reporter strains, no fluorescence was observed in the strains carrying GFP under control of the SAP2 promoter (Fig. 3B). In the control strains expressing GFP from the ADH1 promoter fluorescence of yeast and hyphal cells was seen throughout the course of the infection (data not shown).
In order to analyze SAP4 and SAP5 promoter-dependent GFP reporter gene activation in more detail, between 100 and 200 hyphal cells were counted for each group and time so that the relative percentage of hyphae that had induced SAP4 and SAP5 could be determined. As shown in Table 2, SAP5 promoter activity was detectable on day 1 and remained high (GFP expression in >87% of the hyphae) throughout the course of infection. SAP4 promoter activity was not observed on day 1, and a low level of activity was observed on day 4. There were small increases on days 9 and 14, and by day 28 the number of GFP-expressing cells had increased to 20%. Induction of SAP4 and SAP5 was observed as GFP production in all segments of the hyphal filament. This suggests that inducing signals reached every segment and that every segment was capable of responding to inducing signals from the environment
DISCUSSION
In contrast to previous studies in which methods such as RT-PCR and infections involving knockout organisms were used, we used IVET to examine the SAP expression pattern in a mouse model of vaginal candidiasis. This allowed us to detect transient gene expression on a single-cell basis. SAP5 was induced in 48% ± 10% of fungal cells as early as 1 day after infection, and this was followed by SAP4 expression in 25% ± 10% of the fungal cells as early as day 4. The induction of SAP5 expression reported here was also seen after inoculation in the peritoneal cavity and so is not specific for vaginal infection. In the esophageal epithelium, the SAP5 and SAP6 genes were expressed, but SAP4 was not expressed. This suggests that SAP6 may play a specific role in esophageal mucosa infection and that SAP4 may play a specific role in vaginal infection (44).
None of the other SAP genes tested were induced in our vaginal infection model. The results were in excellent agreement with those obtained by using GFP as a reporter gene. A similar high level of induction of SAP5 was observed after 1 day of infection, and this was followed by detection of SAP4 promoter activity after 4 days of infection. The apparent discrepancies between the percentages of colonies that were MPA sensitive and the numbers of cells that expressed GFP can mostly be explained by the cumulative nature of the recombination-based reporter system. Once a cell has induced the promoter being studied above a threshold level, the MPAR marker is excised and lost irreversibly. These cells and their progeny can no longer revert phenotype, which results in a cumulative induction effect (47). Additionally, the formation of mixed aggregates consisting of sensitive and resistant cells results in an MPA-resistant CFU. This bias may explain the notable variability of our results for different animals. The early pattern of transient expression of SAP4 may indicate that induction of this gene requires strong environmental signals which might not be available for every cell soon after infection.
Induction of SAP4 and SAP5 during infection has been shown to occur in other models of candidiasis by using IVET and conventional RT-PCR. However, induction of SAP expression is thought to be a niche-specific phenomenon. With respect to vaginal infections, in vitro up-regulation of SAP4 and SAP5 after exposure of C. albicans to cultured vaginal epithelial cells has been reported previously (34). The up-regulation of these aspartic proteinase genes coincided with significant epithelial damage. Additionally, a comparative study of isolates collected from patients colonized with C. albicans, as well as isolates collected from sites of active infection, revealed expression of SAP4 and SAP5 in both commensal and infectious isolates (30). The fact that we observed gradual induction of SAP4 and SAP5 during acute infection in our model of vaginal candidiasis suggests that the proteinases are induced as a consequence of infection, which is in contrast to the suggested constitutive SAP4 and SAP5 expression in C. albicans isolates from carriers and infected individuals (30). Strain variability could be responsible for these differences, since variability in SAP expression has also been found among individual patients (30). Alternatively, differences in the technologies employed could account for differences in the results. RT-PCR should effectively detect SAP4 mRNA in a single cell from a pool of Candida cells and should not give the cumulative SAP expression profile that is induced by a majority of cells that are collected from a patient or an animal after experimental infection. Furthermore, collection of isolates from patients does not allow the investigator to observe the initial phases of an infection or colonization like an infection model does. With our approach, we were able to establish through cumulative kinetic analysis which SAP genes were induced after the onset of infection.
The apparent lack of induction of other SAP genes in our in vivo model of candidiasis is in contrast to the findings of other studies in which the workers examined the SAP expression profiles elicited during vaginal candidiasis. RT-PCR-based studies revealed induction of not only SAP4 and SAP5 but also other SAP genes. Specifically, SAP1, SAP2, SAP6, SAP7, SAP9, and SAP10 were found to be induced in response to infection by using the reconstituted human vaginal epithelium model (34), and SAP1 to SAP3 and SAP6 to SAP8 were found to be induced in patient isolates (30). The expression levels of these other SAP genes might be below the detection limit of the FLP and GFP reporter systems. However, it should be noted that significant expression of SAP2 and SAP6 was previously detected in other infection models by using IVET (47), demonstrating that in some host niches expression of these genes is induced at significantly higher levels than the levels observed during infection of the mouse vagina. The relevance of gene expression at levels detectable only by sensitive PCR technology has not been established. A single-cell analysis of SAP genes that are induced by the majority of Candida cells at an active site of infection is more indicative of the SAP expression profile during infection than gene expression in a pool of heterogeneous cells is.
The importance of individual SAP genes for infection has been studied by other researchers by using specific knockout mutants. By using the in vitro reconstituted human vaginal epithelium model, it has been shown that null mutants lacking SAP1 and SAP2 have a drastically reduced potential to cause serious tissue damage (34). Furthermore, SAP2 null mutants exhibited attenuated virulence in the rat model of vaginal candidiasis (5). The discrepancies between our results and those obtained in these studies may be due to basic differences in the model systems (e.g., the use of ovariectomized estrogen-treated rats or estrogen-treated mice). Of particular interest are differences in the physiological pH values of rat, human, and mouse vaginas. The rat and human vaginal pH is around 4.5, whereas the mouse vaginal pH is between 6.2 and 6.5 (6, 24). Since it has been shown that Sap1p to Sap3p have the highest levels of activity at lower pH values and since Sap4p to Sap6p have the highest levels of activity at higher pH values (29), we postulate that the C. albicans gene expression profile depends on the physiological characteristics of the animal model system. However, both systems are readily used to examine Candida vaginitis, and it remains to be seen which model system is more indicative of human vaginal candidiasis. Although the discrepancies between our results and those of other workers may indicate that the mouse model does not adequately reflect the human patient, it should be considered that human vaginal infections are accompanied by elevated pH values.
To investigate the correlation between hyphal formation and SAP activation, we focused on the purported link between SAP5 induction and morphogenic development. Several studies have reported expression of the SAP4 to SAP6 subfamily in response to hyphal formation in vitro (20, 36, 37, 50), but it has also been demonstrated that SAP5 expression still occurs in response to inducing signals when filamentation is blocked (25, 46). Because of the early induction of SAP5 that was observed in our Candida vaginitis model, we investigated how this induction correlated with hyphal development in the initial stages of infection. In our infection model, SAP5 expression was detected only in samples in which the majority of cells had switched to the hyphal growth mode, in contrast to observations made in a model of Candida peritonitis in which significant SAP5 induction occurred before germ tubes were detected (46). Hyphal formation during vaginal infection, however, was not always followed by SAP4 or SAP5 expression, since not all lavage fluids that contained a majority of hyphal cells yielded significant numbers of MPA-sensitive colonies and not all hyphal cells of the GFP reporter strains exhibited fluorescence. Therefore, SAP4 and SAP5 expression depends on signals that also induce hyphal formation in this infection model, but expression of the hypha-associated SAP genes may require additional, consecutive, or stronger signals than hyphal formation requires. Further support for this view comes from the specific kinetics of the observed activation of the SAP4 promoter, which did not immediately follow hyphal formation or SAP5 induction but rather steadily increased throughout the course of the infection.
In this paper, we report the exclusive and differential induction of two of six members of the SAP gene family in a mouse model of vaginal candidiasis. Due to the strong induction of these two SAP genes, we hypothesize that they play an important role in initiation and establishment of infection. We also report differential, gene-specific kinetics of in vivo SAP4 and SAP5 induction, which was associated with hyphal formation at the site of infection. Our results demonstrate that a limited, niche-specific SAP expression pattern is induced during infection in the mouse model of vaginal candidiasis.
ACKNOWLEDGMENTS
We thank B. Bodendorfer and H. Arnold for skillful technical assistance.
This work was supported by Deutsche Forschungsgemeinschaft grants Schr 450/4-1 (to K.S.) and Mo 846/1-3 (to J.M.), by the Interdisciplinary Research Center (IZKF) at the University of Erlangen (grant TP.A15/A3 to B.N.T. and M.S.), and by the NRC-HGF Science and Technology Fund (grant 01SF0201/2.2.).
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Institut für Molekulare Infektionsbiologie, Universitt Würzburg, Würzburg, Germany
ABSTRACT
Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.
INTRODUCTION
Candida albicans is a common commensal fungal organism that is part of the normal microflora of the genital and gastrointestinal tract of healthy individuals. Under appropriate conditions, C. albicans can become an opportunistic pathogen that has debilitating and sometimes deadly consequences for its host (8). One common manifestation of Candida infection is superficial candidiasis, which is a significant problem encountered in clinics worldwide. The most common superficial infections are vaginal candidiasis and mucosal candidiasis of the oropharyngeal cavity and the esophagus (reviewed in reference 3).
Vulvovaginal candidiasis (VVC) affects an estimated three of four women at least once during their reproductive years, and by the age of 25 one-half of all college women will have had at least one physician-diagnosed episode (12, 13, 22, 39). Although some women never contract VVC and others experience only sporadic acute episodes, a third significant subpopulation comprising as many as 5% of all adult women experience subsequent recurrent VVC (10, 31). Although several exogenous factors are thought to increase the incidence of VVC (pregnancy, oral contraceptives, diabetes mellitus, and antibiotics) (38, 40), the pathogenesis of recurrent VVC is still largely unknown.
Several factors of C. albicans were previously identified as virulence determinants; these factors include hyphal formation (41), phenotypic switching (17, 43, 49), and secretion of hydrolytic enzymes (14, 19, 32). Although hyphal formation is probably the best-documented virulence determinant of C. albicans, the production of hydrolytic enzymes, specifically the secreted aspartic proteinases (Saps), as key virulence determinants has been comprehensively studied (4). Ten SAP isogenes encode the Sap proteins (7, 26, 27). The purported functions of Saps during infection include the digestion of host proteins for nutrient supply, evasion of host defenses by degradation of immunoglobulins and complement proteins, adherence, and degradation of host barriers during invasion (19, 20, 23, 35, 44, 50). Due to their wide substrate specificity and broad pH range (2), it is accepted that Saps contribute to the development of active Candida infections.
Previous studies have highlighted the importance of certain SAP genes in relation to various models of infection (21, 29). Furthermore, individual members of the gene family might have their own unique roles in the infectious process because environmental conditions have been shown to affect production of Saps differentially, resulting in a niche-specific expression pattern of SAP genes (14, 16, 18-20, 23, 35, 44, 48, 50). An in vivo expression technology (IVET) that is based on the irreversible deletion of a mycophenolic acid resistance (MPAR) marker from the genome as a stable reporter of gene activation has proven to be very useful for detecting, on a single-cell basis, the induction of genes that are only transiently expressed at a certain stage of infection. By using this system, differential activation of individual members of the SAP gene family was observed in esophageal infections, in intraperitoneal infections, in gastrointestinal infections, and after hematogenous dissemination to the kidneys (23, 44).
Studies investigating the production of SAP mRNA and the virulence of SAP knockout strains during Candida vaginitis have been performed previously. By using an in vitro reconstituted human vaginal epithelium model, detection of SAP1, SAP4, and SAP5 was found to coincide with the onset of significant tissue damage (34). Additional studies have documented the expression of SAP1, SAP3, and SAP6 to SAP8 during human vaginal infection by using reverse transcription (RT)-PCR. In a rat model of Candida vaginitis, the importance of SAP1 to SAP3 was demonstrated by using specific knockout strains of C. albicans (5, 30).
In this study, we examined the SAP expression profile that is exhibited by C. albicans in an experimental mouse model of vaginal candidiasis and how this expression profile correlates with hyphal formation. We used two different genetic reporter systems to detect the activation of individual SAP promoters by host signals, the recombination-based IVET system and C. albicans strains expressing the GFP reporter gene under control of the promoters of selected SAP genes. This allowed us to microanalyze the temporal and spatial expression pattern of SAP genes in single hyphal segments and to test our hypothesis that a specific SAP expression profile is also elicited in this host niche. To our knowledge, this is the first study in which reporter strains were used to analyze both SAP induction and its relationship to hyphal formation in an in vivo model of vaginal candidiasis.
MATERIALS AND METHODS
Strains and growth conditions. All strains were maintained on complete supplement medium without uridine (CSM-ura) agar plates containing (per liter) 6.7 g of yeast nitrogen base without amino acids (Bio 101), 2 g of glucose, and 0.77 g of CSM-ura (Bio 101). For in vivo studies, C. albicans strains were grown to the stationary phase in CSM-ura liquid medium for 16 to 18 h at 28°C on an orbital shaker. After two washes with sterile water, the number of blastoconidia was determined by using a hemocytometer, and blastoconidia were transferred with pipettes directly into the vaginal canal.
The C. albicans strains used in this study and, for clarity, the relevant parent strains are described in Table 1. The C. albicans IVET system has been described previously (44, 47). Briefly, the reporter strains contain the ecaFLP gene, which encodes the site-specific recombinase FLP, under the control of one of the SAP promoters at the original genomic locus and a genomically integrated MPAR marker that is flanked by direct repeats of the FLP recognition sequence (FRT). Induction of the SAP promoter results in excision of the MPAR marker from the genome, so that the corresponding cell and its progeny grow more slowly and form smaller colonies than their parent on agar plates containing a suitable concentration of mycophenolic acid (MPA) (1 μg/ml). For each of the SAP genes (SAP1 to SAP6), two independently generated reporter strains were used which carried the reporter fusion in one of the two possible alleles (e.g., SAP2-1 and SAP2-2) whenever the alleles could be distinguished by restriction site polymorphism and by sequencing of the cloned promoters (Table 1). As controls for the MPA-resistant and -sensitive phenotypes, a strain containing a stably integrated MPAR marker (S2UI1A) and a strain that did not contain the MPAR marker (S2FI1AS) were used. In all in vivo experiments, appropriate dilutions of the inocula were plated on MPA indicator plates to determine the proportion of MPA-sensitive cells before infection, which was always less than 3%. To detect SAP activation during infection, vaginal lavage fluids obtained at different times after inoculation were spread on MPA indicator plates. After incubation at 30°C for 3 to 4 days, the percentage of small colonies was determined. Because the maximum proportion of small colonies in the inocula was 3%, we defined an arbitrary limit of 9% MPA-sensitive colonies (three times the nonspecific background proportion) as a threshold for significant induction of a specific gene.
In addition, a series of strains which carried the GFP gene as a reporter was generated (Morschhuser, unpublished data). Briefly, a C. albicans-adapted GFP reporter gene (28) was substituted for the ecaFLP gene in the corresponding plasmids. In addition, a similar construct was made in which the GFP gene was placed under control of the strong ADH1 (alcohol dehydrogenase) promoter. The reporter fusions were then integrated into the respective loci of parental strain CAI4 (Table 1). Inocula of GFP-expressing strains were microscopically examined to verify that GFP expression had occurred only in cultures of the control strain harboring the ADH1 promoter-GFP fusion and that no detectable GFP expression had occurred in the cultures with SAP promoter-GFP fusions before infection. Validation of green fluorescent protein (GFP) as a reporter of gene expression in vivo has been extensively demonstrated recently (1).
In vivo infection studies and recovery of vaginal C. albicans. Eight- to ten-week-old female BALB/c mice were obtained from Charles Rivers Breeding Laboratories (Sulzfeld, Germany). To induce pseudoestrus during infection, mice were injected subcutaneously with 0.02 mg of estradiol valerate (Sigma Chemical Co., St. Louis, Mo.) per ml in 0.1 ml of sesame oil 72 h before inoculation with C. albicans (9, 33, 42). Estradiol treatments were continued at weekly intervals thereafter. Infection of the vaginal canal was initiated by inoculating mice intravaginally with 5 x 104 stationary-phase cells of a strain in 20 μl of phosphate-buffered saline (PBS) (9). Mice were either sacrificed or anesthetized, and vaginal lavages were performed at different times following infection. We included controls which showed that the use of live or sacrificed mice for vaginal lavage did not affect the number of CFU obtained or the outcome of the infection.
For experiments involving FLP reporter strains, mice were anesthetized and lavaged with 50 μl of sterile PBS on days 1, 4, 9, 14, 20, 27, and 32. Lavage fluids were serially diluted and plated, and the percentage of MPA-sensitive cells was determined (total number of MPA-sensitive cells/total number of CFU x 100). Experiments were repeated at least twice with independently generated reporter strains for each SAP gene.
Studies comparing the induction of SAP5 with the number of CFU, hyphal formation, and hyphal length were performed as follows. Mice were sacrificed 1, 2, 4, 6, 8, 12, 23, 36, and 50 h postinfection and lavaged with 100 μl of sterile PBS. Fifty microliters of lavage fluid was serially diluted and plated, and the total number of CFU and the percentage of MPA-sensitive cells were determined. The remaining vaginal lavage fluid was subjected to microscopic examination as described below.
For experiments involving GFP reporter strains, mice were anesthetized and lavaged with 50 μl of sterile PBS on days 1, 4, 9, 14, 21, and 28. Immediately following each lavage, samples were subjected to microscopic examination as described below. Experiments were repeated with two independently generated reporter strains for each SAP gene.
Microscopic examination. For microscopic examination of the cellular morphology of C. albicans FLP reporter strains during infection, vaginal lavage samples from sacrificed mice were solubilized by using a 20% KOH solution at room temperature. This step was included because of the viscosity of the samples due to the presence of large amounts of mucus in the vagina. Specimens were centrifuged (1,500 x g for 10 min), and the sediment was mounted on glass slides in the presence of fluorescent brighteners (Calcofluor white; Sigma) for epifluorescence microscopy (Zeiss Axiophot). Approximately 100 cells from multiple slides were observed to determine the percentages of hyphae and blastospores in the vaginal lavage fluid. Images of representative fields were acquired with a SPOT camera, and measurements were taken with the MetaView software package to determine the total hyphal length and the length of the individual hyphal segments.
GFP expression was examined directly following vaginal lavage of anesthetized mice. Thin wet-mount slides (each containing 5 μl of the vaginal lavage fluid) were prepared. Between 100 and 200 individual cells, depending on the vaginal fungal burden, were evaluated for GFP expression by epifluorescence microscopy. Representative images for conventional phase-contrast microscopy and consecutive fluorescence exposures of identical fields were acquired with the MetaView software package. Overlays created by merging the two images are presented below.
Statistical analysis. The unpaired Student t test was used to analyze the data when applicable. Significant differences were defined as a confidence level at which the P value was <0.05. Unless indicated otherwise, data are expressed as means ± standard errors of the means. Correlations between increases in average hyphal length either for time after infection or for increases in the average percentage of MPA-sensitive cells were analyzed by using linear regression by minimizing the sum of squares.
RESULTS
Analysis of SAP expression in vaginal candidiasis. The recombination-based IVET is very useful for measuring cumulative gene expression because cells that have lost the MPAR marker by FLP-mediated recombination after induction of the target gene promoter remain MPA sensitive even when the promoter is down-regulated again at later stages of the infection. We determined the in vivo SAP expression profile of C. albicans that is elicited during a vaginal infection on a single-CFU basis. After induction of pseudoestrus, the mice were infected with SAP1 to SAP6 reporter strains and two controls (see Materials and Methods). Mice were lavaged on days 1, 4, 9, 14, 20, 27, and 32, and the lavage fluids were plated to determine the percentage of MPA-sensitive colonies.
Figure 1 shows the results of two experiments for three representative days of the 32-day experiment (days 4, 14, and 27). By day 4 postinfection, the SAP5 promoter had been detectably activated in approximately 100% of the cells recovered from the vagina. In fact, SAP5 induction was observed in 48% ± 10% of the cells as early as day 1 after infection. SAP4 activation was also detected early in the infection, and there were only slight differences in the two clones tested. By day 4 postinfection, we detected induction of SAP4 in 25% ± 10% of the C. albicans cells recovered from vaginal lavage fluids. By day 14 postinfection, all mice infected with the SAP4 or SAP5 reporter strains contained significant proportions of MPA-sensitive cells (Fig. 1B), and by day 27 after infection 100% of the colonies from these mice were MPA sensitive (Fig. 1C). Surprisingly, in contrast to findings of other studies in which vaginal infections were evaluated, no other SAP genes examined were highly or consistently induced at detectable levels over the course of the 32-day vaginal infection.
SAP5 expression occurs after a matter of hours and correlates with hyphal formation. Since we observed such high and early activation of SAP5, we investigated the onset of induction by examining earlier times during infection, starting 1 h postinoculation. Lavage fluids were plated, and a portion of each preparation was used for microscopic examination of morphogenic development. In this way, we assessed if SAP5 expression correlated with the fungal load and/or hyphal formation in the vagina.
To address the first point, we determined the average number of CFU obtained from three mice per time and plotted the average number of CFU against the rise in the number of MPA-sensitive colonies. As illustrated in Fig. 2A, there was an initial drop in the number of CFU following inoculation, but thereafter the total number of CFU grown from the vaginal lavage fluid remained relatively constant throughout the 50 h of observation (between 2.3 x 103 ± 1.4 x 103 and 1.1 x 104 ± 0.6 x 104 CFU). However, the percentage of MPA-sensitive colonies increased in some mice (Fig. 2B) to 17% as early as 2 h after infection, and the mean level of SAP5 induction rose sharply after 12 h (significance for the group of animals reached between 24 and 36 h, P < 0.03).
With regard to the second point, we examined the correlation between hyphal formation and SAP5 induction by comparing the proportion of MPA-sensitive colonies with the morphology of the cells in the samples. As shown in Fig. 2B, expression of SAP5 was variable. In some animals, it was detectable after 2 h of infection, and expression was reproducibly detected between 12 and 23 h. After examination of the lavage fluids for the presence of hyphae (Fig. 2C), we found that substantial SAP5 activation was detected only in lavage samples in which the majority of cells were in the hyphal form, but it must be emphasized that not all samples with significant amounts of hyphal cells gave rise to MPA-sensitive colonies, because SAP5 expression was not observed in every sample in which hyphae predominated over yeast cells. For example, after 2 h of infection the mouse 1 (M1) sample contained 78% hyphae, and 17% of the colonies were MPA sensitive, well above the background values. In contrast, strong hyphal induction (71% hyphae) was also observed in the M2 sample after 4 h of infection, but only 3% MPA-sensitive colonies were detected in this sample, a value which is similar to the background values in the inocula before infection. None of the samples containing a majority of blastospores (M1 sample at 1 h; M2 and M3 samples at 2 h; M3 sample at 4 h; M1 sample at 6 and 12 h) gave rise to a considerable number of MPA-sensitive colonies, indicating that the level of SAP5 induction was below the detection limit of the reporter system in these samples.
SAP5 induction correlated with the presence of hyphae but not with hyphal length. The mean hyphal length increased constantly over time, indicating that there was a constant rate of growth and propulsion of filaments (r2 = 0.91, P 0.03). However, the increase in the number of MPA-sensitive cells did not correlate with hyphal length (r2 = 0.19, P > 0.05).
Detection of SAP expression during vaginal infection by using the GFP reporter gene. In order to confirm the results obtained with the FLP reporter strains, we tested if GFP could be used as an alternative reporter of gene expression in the mouse model of Candida vaginitis. Preliminary experiments were performed with strains that constitutively expressed GFP from the ADH1 promoter. As shown in Fig. 3A, even in the presence of cellular infiltration and large amounts of mucus distinct hyphal cells were distinguishable from background fluorescence, and a detailed hyphal structure could be observed. These results suggested that GFP is a suitable reporter for analysis of gene expression in this infection model and that it could even be used to detect possible differences in SAP induction in individual hyphal compartments of the same filament.
We then investigated the expression of SAP4 and SAP5 during vaginal infection using C. albicans strains that carried chromosomally integrated SAP-GFP fusions (see Table 1 for strain descriptions). Since we were unable to detect expression of the SAP2 gene in this infection model with the recombination-based IVET, we also included two reporter strains in which GFP was expressed from the promoters of either of the two SAP2 alleles, which have been shown to differ in their inducibility in this strain background due to allelic differences (45).
As shown in Fig. 3B, strong SAP5 promoter activity was observed as early as day 1 and persisted through day 28 following infection. SAP4 expression was detected starting on day 4 postinfection and was present through day 28. In contrast, and in agreement with the results obtained with the FLP reporter strains, no fluorescence was observed in the strains carrying GFP under control of the SAP2 promoter (Fig. 3B). In the control strains expressing GFP from the ADH1 promoter fluorescence of yeast and hyphal cells was seen throughout the course of the infection (data not shown).
In order to analyze SAP4 and SAP5 promoter-dependent GFP reporter gene activation in more detail, between 100 and 200 hyphal cells were counted for each group and time so that the relative percentage of hyphae that had induced SAP4 and SAP5 could be determined. As shown in Table 2, SAP5 promoter activity was detectable on day 1 and remained high (GFP expression in >87% of the hyphae) throughout the course of infection. SAP4 promoter activity was not observed on day 1, and a low level of activity was observed on day 4. There were small increases on days 9 and 14, and by day 28 the number of GFP-expressing cells had increased to 20%. Induction of SAP4 and SAP5 was observed as GFP production in all segments of the hyphal filament. This suggests that inducing signals reached every segment and that every segment was capable of responding to inducing signals from the environment
DISCUSSION
In contrast to previous studies in which methods such as RT-PCR and infections involving knockout organisms were used, we used IVET to examine the SAP expression pattern in a mouse model of vaginal candidiasis. This allowed us to detect transient gene expression on a single-cell basis. SAP5 was induced in 48% ± 10% of fungal cells as early as 1 day after infection, and this was followed by SAP4 expression in 25% ± 10% of the fungal cells as early as day 4. The induction of SAP5 expression reported here was also seen after inoculation in the peritoneal cavity and so is not specific for vaginal infection. In the esophageal epithelium, the SAP5 and SAP6 genes were expressed, but SAP4 was not expressed. This suggests that SAP6 may play a specific role in esophageal mucosa infection and that SAP4 may play a specific role in vaginal infection (44).
None of the other SAP genes tested were induced in our vaginal infection model. The results were in excellent agreement with those obtained by using GFP as a reporter gene. A similar high level of induction of SAP5 was observed after 1 day of infection, and this was followed by detection of SAP4 promoter activity after 4 days of infection. The apparent discrepancies between the percentages of colonies that were MPA sensitive and the numbers of cells that expressed GFP can mostly be explained by the cumulative nature of the recombination-based reporter system. Once a cell has induced the promoter being studied above a threshold level, the MPAR marker is excised and lost irreversibly. These cells and their progeny can no longer revert phenotype, which results in a cumulative induction effect (47). Additionally, the formation of mixed aggregates consisting of sensitive and resistant cells results in an MPA-resistant CFU. This bias may explain the notable variability of our results for different animals. The early pattern of transient expression of SAP4 may indicate that induction of this gene requires strong environmental signals which might not be available for every cell soon after infection.
Induction of SAP4 and SAP5 during infection has been shown to occur in other models of candidiasis by using IVET and conventional RT-PCR. However, induction of SAP expression is thought to be a niche-specific phenomenon. With respect to vaginal infections, in vitro up-regulation of SAP4 and SAP5 after exposure of C. albicans to cultured vaginal epithelial cells has been reported previously (34). The up-regulation of these aspartic proteinase genes coincided with significant epithelial damage. Additionally, a comparative study of isolates collected from patients colonized with C. albicans, as well as isolates collected from sites of active infection, revealed expression of SAP4 and SAP5 in both commensal and infectious isolates (30). The fact that we observed gradual induction of SAP4 and SAP5 during acute infection in our model of vaginal candidiasis suggests that the proteinases are induced as a consequence of infection, which is in contrast to the suggested constitutive SAP4 and SAP5 expression in C. albicans isolates from carriers and infected individuals (30). Strain variability could be responsible for these differences, since variability in SAP expression has also been found among individual patients (30). Alternatively, differences in the technologies employed could account for differences in the results. RT-PCR should effectively detect SAP4 mRNA in a single cell from a pool of Candida cells and should not give the cumulative SAP expression profile that is induced by a majority of cells that are collected from a patient or an animal after experimental infection. Furthermore, collection of isolates from patients does not allow the investigator to observe the initial phases of an infection or colonization like an infection model does. With our approach, we were able to establish through cumulative kinetic analysis which SAP genes were induced after the onset of infection.
The apparent lack of induction of other SAP genes in our in vivo model of candidiasis is in contrast to the findings of other studies in which the workers examined the SAP expression profiles elicited during vaginal candidiasis. RT-PCR-based studies revealed induction of not only SAP4 and SAP5 but also other SAP genes. Specifically, SAP1, SAP2, SAP6, SAP7, SAP9, and SAP10 were found to be induced in response to infection by using the reconstituted human vaginal epithelium model (34), and SAP1 to SAP3 and SAP6 to SAP8 were found to be induced in patient isolates (30). The expression levels of these other SAP genes might be below the detection limit of the FLP and GFP reporter systems. However, it should be noted that significant expression of SAP2 and SAP6 was previously detected in other infection models by using IVET (47), demonstrating that in some host niches expression of these genes is induced at significantly higher levels than the levels observed during infection of the mouse vagina. The relevance of gene expression at levels detectable only by sensitive PCR technology has not been established. A single-cell analysis of SAP genes that are induced by the majority of Candida cells at an active site of infection is more indicative of the SAP expression profile during infection than gene expression in a pool of heterogeneous cells is.
The importance of individual SAP genes for infection has been studied by other researchers by using specific knockout mutants. By using the in vitro reconstituted human vaginal epithelium model, it has been shown that null mutants lacking SAP1 and SAP2 have a drastically reduced potential to cause serious tissue damage (34). Furthermore, SAP2 null mutants exhibited attenuated virulence in the rat model of vaginal candidiasis (5). The discrepancies between our results and those obtained in these studies may be due to basic differences in the model systems (e.g., the use of ovariectomized estrogen-treated rats or estrogen-treated mice). Of particular interest are differences in the physiological pH values of rat, human, and mouse vaginas. The rat and human vaginal pH is around 4.5, whereas the mouse vaginal pH is between 6.2 and 6.5 (6, 24). Since it has been shown that Sap1p to Sap3p have the highest levels of activity at lower pH values and since Sap4p to Sap6p have the highest levels of activity at higher pH values (29), we postulate that the C. albicans gene expression profile depends on the physiological characteristics of the animal model system. However, both systems are readily used to examine Candida vaginitis, and it remains to be seen which model system is more indicative of human vaginal candidiasis. Although the discrepancies between our results and those of other workers may indicate that the mouse model does not adequately reflect the human patient, it should be considered that human vaginal infections are accompanied by elevated pH values.
To investigate the correlation between hyphal formation and SAP activation, we focused on the purported link between SAP5 induction and morphogenic development. Several studies have reported expression of the SAP4 to SAP6 subfamily in response to hyphal formation in vitro (20, 36, 37, 50), but it has also been demonstrated that SAP5 expression still occurs in response to inducing signals when filamentation is blocked (25, 46). Because of the early induction of SAP5 that was observed in our Candida vaginitis model, we investigated how this induction correlated with hyphal development in the initial stages of infection. In our infection model, SAP5 expression was detected only in samples in which the majority of cells had switched to the hyphal growth mode, in contrast to observations made in a model of Candida peritonitis in which significant SAP5 induction occurred before germ tubes were detected (46). Hyphal formation during vaginal infection, however, was not always followed by SAP4 or SAP5 expression, since not all lavage fluids that contained a majority of hyphal cells yielded significant numbers of MPA-sensitive colonies and not all hyphal cells of the GFP reporter strains exhibited fluorescence. Therefore, SAP4 and SAP5 expression depends on signals that also induce hyphal formation in this infection model, but expression of the hypha-associated SAP genes may require additional, consecutive, or stronger signals than hyphal formation requires. Further support for this view comes from the specific kinetics of the observed activation of the SAP4 promoter, which did not immediately follow hyphal formation or SAP5 induction but rather steadily increased throughout the course of the infection.
In this paper, we report the exclusive and differential induction of two of six members of the SAP gene family in a mouse model of vaginal candidiasis. Due to the strong induction of these two SAP genes, we hypothesize that they play an important role in initiation and establishment of infection. We also report differential, gene-specific kinetics of in vivo SAP4 and SAP5 induction, which was associated with hyphal formation at the site of infection. Our results demonstrate that a limited, niche-specific SAP expression pattern is induced during infection in the mouse model of vaginal candidiasis.
ACKNOWLEDGMENTS
We thank B. Bodendorfer and H. Arnold for skillful technical assistance.
This work was supported by Deutsche Forschungsgemeinschaft grants Schr 450/4-1 (to K.S.) and Mo 846/1-3 (to J.M.), by the Interdisciplinary Research Center (IZKF) at the University of Erlangen (grant TP.A15/A3 to B.N.T. and M.S.), and by the NRC-HGF Science and Technology Fund (grant 01SF0201/2.2.).
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