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Reduced expression of the hyphal-independent Candida albicans proteinase genes SAP1 and SAP3 in the efg1 mutant is associated with attenuated virulence during infection of oral epithelium

^1,3Department of Dermatology and Allergology¹ and Department of Parodontology³, University of Munich, Munich, Germany ²Robert Koch-Institut, Berlin, Germany

Correspondence Martin Schaller Martin.Schaller{at}lrz.uni- muenchen.de

Received November 15, 2002
Accepted March 31, 2003

	Abstract

TOP Abstract INTRODUCTION METHODS RESULTS DISCUSSION Acknowledgements REFERENCES

 
The transition of Candida albicans from a yeast to a hyphal form is controlled by several transcriptional factors, including the key regulators Cph1 and Efg1, and is considered an important virulence attribute. These factors, especially Efg1, regulate the expression of hyphal-associated genes e.g. SAP4–SAP6. In order to investigate the relevance of these transcriptional regulators for hyphal-independent SAP genes, recently constructed cph1 and efg1 single mutants and a cph1/efg1 double mutant lacking these factors were tested during interaction with oral epithelium and polymorphonuclear neutrophils. In contrast to the parental wild-type strain and the cph1 mutant, the efg1 and the cph1/efg1 mutants did not produce hyphal forms in all experiments and were less capable of damaging epithelial cells and neutrophil granulocytes. The attenuated epithelial lesions of these mutants were correlated not only with reduced expression of the hyphal-associated gene SAP4, but also with the lack of SAP1 and SAP3 expression previously shown to be important for oral infections. An efg1 mutant strain carrying a plasmid-borne copy of the EFG1 gene regained hyphal growth, damage of keratinocytes, granulocytes and the expression of SAP1 and SAP3. Although efg1 and cph1/efg1 mutants did not produce germ tubes during infection, expression of the hyphal-associated genes SAP5 and SAP6 was not completely abolished. A reduced capacity to stimulate an epithelial immune response manifested by a delayed onset of IL-1ß, IL-8 and TNF expression was only observed in the cph1/efg1-infected tissue. These results provide further evidence for a combined regulation of different virulence factors, such as dimorphism and expression of SAP genes. Furthermore, it could be demonstrated that the lack of Efg1 also caused reduced expression of hyphal-independent SAP genes. Both the EFG1 and the CPH1 gene products are necessary for adequate induction of an immune response.

	INTRODUCTION

TOP Abstract INTRODUCTION METHODS RESULTS DISCUSSION Acknowledgements REFERENCES

 
The yeast-to-hyphal transition (dimorphism) is known to be one of the most important virulence factors of the fungal pathogen Candida albicans (Brown & Gow, 1999; Calderone & Fonzi, 2001; Ernst, 2000). Dimorphism is regulated by different cross-talking signal transduction pathways. Efg1 (Stoldt et al., 1997; Bockmuhl & Ernst, 2001) and Cph1 (Bockmuhl & Ernst, 2001) have been identified as key transcriptional regulators for the cAMP-protein kinase (PKA) and the mitogen-activated protein kinase (MAPK) pathway, respectively. The role of these factors in morphogenesis and their relevance for Candida infections was studied using strains which lacked either functional Efg1, Cph1 or both factors (Brown & Gow, 1999; Stoldt et al., 1997; Bockmuhl & Ernst, 2001; Dieterich et al., 2002; Kohler & Fink, 1996; Leberer et al., 1997; Lo et al., 1997; Sonneborn et al., 1999b; Weide & Ernst, 1999; Lewis et al., 2002; Phan et al., 2000). The results obtained with these mutants suggested that disruption of CPH1 caused attenuated hyphal formation abilities on solid media while mutants lacking EFG1 failed to produce hyphal cells under most conditions investigated. Furthermore, mutants lacking EFG1 showed a significantly attenuated virulence phenotype in a mouse model of systemic infection (Lo et al., 1997), in the interaction with macrophages (Lo et al., 1997), in the ability to colonize successfully on polyurethane central venous catheters (Lewis et al., 2002) and in the capacity to invade or injure endothelial (Phan et al., 2000), epidermal or intestinal (Dieterich et al., 2002) cells. However, under laboratory conditions of microaerophilic growth (Sonneborn et al., 1999a) and using an alternative animal model (Riggle et al., 1999), the cph1/efg1 double mutant was still able to form filaments and to produce invasive lesions in the tongue of the infected germ-free piglets (Riggle et al., 1999).

For oral candidosis, epithelial cells and polymorphonuclear neutrophils (PMNs) are the first line of host defence (Eversole et al., 1997; Challacombe, 1994; Farah et al., 2001). We therefore investigated the interaction of the cph1, efg1 and cph1/efg1 mutants, their parental wild-type strain SC5314 and an EFG1 revertant with reconstituted human oral epithelium (RHE) and with PMNs. Previously we have shown that certain secreted aspartyl proteinases (Saps) contribute to virulence in this model (Schaller et al., 1998, 1999) and that the mucosal immune response is modulated differentially by different Candida species depending on their virulence potential (Schaller et al., 2002). Recently it has been shown that Efg1 is involved in the regulation of both hyphal formation and expression of hyphal-associated SAP genes (Schröppel et al., 2000; Staib et al., 2002; Felk et al., 2002). In the present study, we focused on the consequence of EFG1 and CPH1 disruption on the expression of hyphal-independent SAP genes and the induction of the immunomodulatory response by the hyphal-deficient mutants during infection of oral epithelium in comparison with wild-type cells.

	METHODS

TOP Abstract INTRODUCTION METHODS RESULTS DISCUSSION Acknowledgements REFERENCES

 
Candida strains, culture media and growth conditions.

For this study, the clinical C. albicans wild-type isolate designated SC5314 (Gillum et al., 1984), the single mutants cph1 (Lo et al., 1997) and efg1 (Stoldt et al., 1997), the double mutant cph1/efg1 (Lo et al., 1997) and an EFG1 reconstituted strain (Lo et al., 1997) were used. For the incubation of C. albicans with PMNs and for the infection of the reconstituted epithelium, inocula were prepared as reported previously (Schaller et al., 1998, 1999).

Reconstituted human epithelium.

The reconstituted human epithelium was supplied by Skinethic Laboratory (Nice, France). Epithelial cells were seeded on inert filter substrates to form multi-layered oral epithelium. Cultures were incubated in serum-free conditions in a defined medium based on the MCDB-153 medium (Clonetics). This fully defined nutrient medium feeds the basal cells through the filter substratum. The medium was changed every 24 h. Three replicate infection experiments were performed with the C. albicans wild-type SC5314, the single mutants cph1 and efg1, the double mutant cph1/efg1 and the EFG1 revertant. Epithelia were infected with 2x10⁶ C. albicans yeast cells of each strain in 50 µl PBS for 12, 36, 46 and 52 h as described previously (Schaller et al., 1998, 1999). Histological lesions of the samples caused by C. albicans were investigated by light microscopy. The C. albicans-induced epithelial damage was quantified by a lactate dehydrogenase (LDH) release assay, the expression of SAP genes and the epithelial immune response was studied by RT-PCR and the analysis of Sap protein secretion was performed by immunoelectron microscopy (see below).

Light microscopy.

Light microscopical studies were performed as previously described (Schaller et al., 1998, 1999) to evaluate histological changes during infection. A part of each specimen was fixed, postfixed and embedded in glycide ether. The small blocks of tissue were cut using an ultra-microtome (Ultracut; Reichert). Semi-thin sections (1 µm) were studied with a light microscope after staining with 1 % toluidine blue and 1 % pyronine G (Merck). The histological changes of the skin were evaluated on the basis of 50 sections from five different sites for each infected epithelium.

Assay of LDH activity.

The release of LDH from epithelial cells into the surrounding medium was monitored as a measure of epithelial cell damage. LDH release in the maintenance media of the cultures from uninfected and infected epithelial cells was measured at 12 and 36 h. The LDH activity was analysed spectrophotometrically by measuring the NADH disappearance rate at 340 nm as a main wavelength during the LDH-catalysed conversion of pyruvate to lactate according to the Wróblewski-La Due method (Wróblewski & John, 1955). The LDH activity is expressed as U l^-1 at 37 °C.

RNA isolation, cDNA synthesis (RT), PCR and pairs of primers.

RT-PCR was used for analysis of SAP and cytokine gene expression during epithelial infection. Details of RNA preparation and cDNA synthesis have been published previously (Felk et al., 2002). For an internal mRNA control and for detection of genomic DNA, we used EFB1 (Maneu et al., 1996). The DNA amplification fragment contained a 364 bp intron. Absence of genomic DNA was verified by a single PCR product of 564 bp. For detection of EFB1 and SAP1–10 transcripts specific pairs of primers were used (Table 1). The specificity of each set of primers was confirmed using genomic DNA. A similar sensitivity of the primer pairs for DNA amplification was determined by testing dilutions of genomic DNA. The cDNA samples were subjected to 35 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 60 °C, and extension for 1 min at 72 °C, and 10 min at 72 °C (final).

The measurement of epithelial cell cytokine gene expression was studied with specific primers for IL-1ß, IL-8 and TNF (Table 1) by RT-PCR in uninfected RHE and 12, 36, 46 and 52 h after infection with C. albicans parental and mutant strains. Primers for amplification of the constitutive expressed genes encoding aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal controls (Table 1). The PCR conditions were 35 cycles of 1 min at 95 °C, 1 min at 65 °C, 1 min at 72 °C, and 5 min at 72 °C (final).

Immunoelectron microscopy.

Post-embedding immunogold labelling was carried out as previously described (Schaller et al., 1998, 1999) for intracellular detection of Sap antigen in epithelial samples infected with SC5314, cph1, efg1, cph1/efg1 and the EFG1 revertant taken after 12 and 36 h. After fixation, specimens were embedded in LR-White. Sections, 80–100 nm thick, were mounted on nickel grids. Grids were then incubated with anti-Sap polyclonal rabbit antibodies, directed against Sap1–3 (Borg-von Zepelin et al., 1998). After washing overnight with PBS, grids were incubated with 10-nm-gold-conjugated goat anti-rabbit IgG (Auroprobe EM Immunogold reagents; Amersham). Grids were then fixed with 2 % glutaraldehyde and stained with 0.5 % uranyl acetate for 10 min and 2.7 % lead citrate for 5 min (Ultrastainer; LKB) at 20 °C. For examination of gold particles a Zeiss EM 902 transmission electron microscope was used operating at 80 kV, at magnifications between x3000 and x85 000. To back up the data statistically, we counted gold particles of 21 randomly chosen cells from each strain.

Isolation of PMNs.

Neutrophils obtained from normal human volunteers were isolated from heparinized whole blood using the Histopaque-1119 in combination with Histopaque-1077 (Sigma) solution. The cells were suspended at a concentration of 5x10⁷ ml^-1 in RPMI 1640 medium (Sigma) containing 10 % FCS. Residual erythrocytes were removed by hypotonic lysis. Typical morphology of the PMNs was proved after Giemsa staining with light microscopy to ensure that a pure population of neutrophils (>95 % purity) had been isolated. The cells were vital-stained using the trypan blue dye exclusion method. Numbers of vital and non-vital leukocytes per sample were assessed using a Neubauer chamber. Viability of 98 % could be demonstrated.

Incubation of C. albicans with PMNs.

A suspension of 4x10⁷ cells of the wild-type isolate SC5314, the mutants cph1, efg1 and cph1/efg1 and the EFG1 revertant in PBS was incubated for 150 min at 37 °C with 3x10⁶ neutrophils to study the interaction of the different strains with PMNs by conventional electron microscopy and to evaluate the inhibitory effect of PMNs on yeast cell growth by a killing assay (see below).

Conventional electron microscopy.

Electron microscopy was used to study the interaction of SC5314, cph1, efg1, cph1/efg1 and the EFG1 revertant with PMNs. Specimens were fixed in a phosphate-buffered solution (0.05 M, pH 7.3) with 2.5 % glutaraldehyde and 2 % formaldehyde following standard methods. Postfixation was based on 1 % osmium tetroxide in 0.1 M phosphate buffer at pH 7.3 at room temperature; the specimens were embedded in glycide ether. Ultrathin sections, 60–90 nm thick, were mounted on uncoated copper grids and stained in 2 % uranyl acetate for 30 min, then in Reynold's lead citrate for 8 min, and examined using a Zeiss EM 902 transmission electron microscope. To support our data statistically, we counted the number of C. albicans cells ingested by the PMNs and the number of obviously damaged fungal cells and leukocytes for each experiment by analysis of 30 representative electron microscopical figures.

Killing assay.

A killing assay was used to study the inhibitory effect of PMNs on growth of the different C. albicans strains. It was performed by plating the C. albicans parental, mutant and revertant samples 150 min after incubation with and without PMNs on Sabouraud's dextrose agar. Before plating, samples were vigorously agitated by using a Pasteur pipette to separate big clumps of fungal cells into single cells and diluted 1 : 1000 and 1 : 10 000 in PBS. The presence of single cells was proved by light microscopy. Yeast cell viability was determined by assessment of the c.f.u. produced after incubation for 24 h at 37 °C on Sabouraud's dextrose agar. Three independent experiments were performed for each strain with and without PMNs.

Reproducibility and verification of the results.

In order to examine the reproducibility of the histological alterations caused by the C. albicans strains during RHE infection and interaction with PMNs, all experiments were carried out in triplicate.

RT-PCR analysis of SAP gene expression during infection of RHE in vitro was done in duplicate from each sample. Similar levels of cDNA in the experiments were verified by amplification of transcripts from housekeeping genes as internal controls. In all experiments, levels of amplification products for the genes encoding aldolase and GAPDH (keratinocytes) and EFB1 (C. albicans) were identical.

Statistical analysis.

Statistical significance was determined using the Least Significance Difference Test. All comparisons were two-sided and a P value of less than 0.05 was considered significant.

	RESULTS

TOP Abstract INTRODUCTION METHODS RESULTS DISCUSSION Acknowledgements REFERENCES

 
Mutants lacking EFG1 have attenuated abilities to damage epithelial cells during infection of oral epithelium

The virulence phenotype after infection of RHE with the mutants cph1, efg1 and cph1/efg1, the parental wild-type strain and the EFG1 revertant was tested. After 12 h, epithelium inoculated with the parental strain, the mutants and the revertant was not significantly damaged. Thirty-six hours after infection, the efg1 and the cph1/efg1 mutants showed reduced epithelial damage (Fig. 1c, d) as compared with the wild-type (Fig. 1a) and the cph1 infection (Fig. 1b). Plasmid-borne EFG1 expression in the efg1 mutant led to reconstitution of yeast-to-hyphal transition and epithelial lesions (Fig. 1e). These histological differences of the phenotype were confirmed by significant differences of LDH release as a marker of cell injury (Table 2).

View larger version (89K): [in this window] [in a new window]   Fig. 1. Histological damage of RHE and SAP1–10 expression for each of the Candida strains (lanes numbered 1–10) SC5314 (a), cph1 (b), efg1 (c), cph1/efg1 (d) and the EFG1 revertant (e) 36 h after infection. Vacuolization and oedema of all keratinocyte layers is accompanied by invasion of all epithelial layers by SC5314 (a), cph1 (b) and the EFG1 revertant (e). There was slight vacuolization and oedema of the superficial keratinocyte layers after infection of RHE with efg1 (c) and cph1/efg1 (d). Similar levels of cDNA and absence of contaminating genomic DNA in the samples are verified by equal amounts of EFB1 transcripts and by a single PCR product of 564 bp (f).

Since the hyphal-deficient mutants cph1/efg1 and efg1 showed a strongly attenuated virulence phenotype we wondered if this is correlated with an altered SAP expression profile. Therefore, we studied SAP expression of these mutants by RT-PCR 12 and 36 h after epithelial infection and compared the expression pattern with those of the more virulent SC5314 wild-type parental, cph1 mutant and EFG1 revertant strains.

At 12 h, SC5314, cph1 and EFG1 revertant strains expressed transcripts of SAP2, SAP5, SAP6, SAP9 and SAP10 while for cph1/efg1 and efg1 only SAP9 and SAP10 cDNA was detected. These expression patterns were not accompanied by significant epithelial lesions.

Twenty-four hours later, SC5314 and cph1 showed similar epithelial damage and a SAP expression pattern with transcripts for most SAP genes (Fig. 1a, b). In contrast, the reduced virulent phenotype of efg1 and cph1/efg1 mutants at the same time was accompanied by a lack of SAP1, SAP3, SAP4 and SAP7 expression (Fig. 1c, d). The revertant strain carrying a plasmid encoding the EFG1 gene caused increased tissue damage and had the ability to express these SAP genes (Fig. 1e).

As seen in Fig. 1, the number of C. albicans cells was decreased in the samples which were infected with EFG1-deficient mutants. The majority of these cells were separated from the mucosal equivalent during the fixation and embedding process due to a reduced adherence. To rule out lower RNA levels isolated from these samples as the reason for the altered SAP expression profiles, we amplified the cDNA of the C. albicans EFB1 gene (Maneu et al., 1996), which is expressed in living C. albicans cells to a similar extent under all conditions and is therefore a useful internal standard (Schaller et al., 1998, 1999). Similar expression levels of EFB1 relative to the same amount of total RNA used in each RT-PCR experiment suggested that all strains were growing on the RHE tissue (Fig. 1f). A similar growth rate of all strains was confirmed by cell counting and/or microscopical observation of hyphal proliferation.

Mutants lacking EFG1 are more susceptible to PMNs

To study the interaction of the hyphal-deficient mutants with host cells we investigated the resistance of the cph1, efg1 and cph1/efg1 mutants and the EFG1 revertant to phagocytosis by PMNs in comparison with the parental strain (Fig. 2a–e). Normal morphology of C. albicans cells (Fig. 2f) and PMNs (Fig. 2g) after isolation of the cells was demonstrated by electron microscopy before the beginning of the experiments. Typical morphology of the PMNs was further proved after Giemsa staining with light microscopy to ensure that a pure population of neutrophils (>95 % purity) had been isolated. Vitality of the isolated PMNs without incubation with C. albicans was 98 % as monitored by the trypan blue staining method. At the ultrastructural level, 150 min after contact with PMNs, the great majority of wild-type, cph1 mutant and EFG1 revertant cells showed extensive hyphal formation and were localized extracellularly (Fig. 2a, b, e; Table 3). The PMNs that were in contact with these C. albicans cells often showed signs of necrosis (Fig. 2a, b, e; Table 3). In sharp contrast, most of the efg1 and cph1/efg1 cells were ingested by the PMNs (Fig. 2c, d; Table 3). Furthermore, escaping from the neutrophils by forming germ tubes was not observed. The host cells showed no alterations of the morphology while many of the engulfed yeast cells demonstrated signs of severe damage (Fig. 2c, d). Statistical analysis of the number of intra- and extracellularly located C. albicans cells and of damaged fungal and effector cells in the experiments clearly demonstrated an attenuated resistance of cph1/efg1 and efg1 mutants to neutrophil activities compared with the wild-type, cph1 and the EFG1 revertant (Table 3).

View larger version (82K): [in this window] [in a new window]   Fig. 2. Ultrastructural morphology 150 min after co-incubation of SC5314 (a), cph1 (b), efg1 (c), cph1/efg1 (d) and the EFG1 revertant (e) with PMNs, and normal morphology of undamaged C. albicans (f) and PMN (g) cells. The great majority of SC5314 (a), cph1 (b) and EFG1 revertant (e) cells are situated extracellularly and are regularly formed. In contrast, PMNs are highly damaged, demonstrating signs of necrosis (N) as oedema. cph1/efg1 (d) and efg1 (c) cells (*) are mainly intracellular and often highly damaged by healthy PMNs.

The viability of C. albicans parental, mutant and revertant strains was also compared by a killing assay. Cytotoxic effects of PMNs on Candida cells were analysed by a quantitative plate count method. After a defined period of exposure to PMNs, samples were plated on Sabouraud's agar. Controls included samples of these Candida strains without PMNs. After an incubation period of 24 h, growth of all strains was inhibited in the presence of PMNs. Similar inhibition rates were seen for the parental strain, the cph1 mutant and the EFG1 revertant while for efg1 and cph1/efg1 the number of survivors was significantly reduced (Table 3).

Lack of SAP1 and SAP3 expression by cph1/efg1 and efg1 mutants correlates with reduced levels of Sap1–3 protein

In former studies, we demonstrated the importance of Sap1–3 but not of Sap4–6 for virulence in experimental oral candidosis (Schaller et al., 1999). We therefore focused our immunoelectron microscopical studies on Sap1–3 secretion during epithelial infection. As compared with the intensive anti-Sap1–3 labelling of each wild-type cell at 36 h the number of gold particles found within the great majority of the cph1/efg1 and efg1 mutant cell walls was strongly reduced. Plasmid-borne EFG1 expression reconstituted an anti-Sap1–3 labelling of the EFG1 revertant strain similar to wild-type cells (Fig. 3; Table 4).

View larger version (98K): [in this window] [in a new window]   Fig. 3. Ultrastructural gold labelling of SC5314 (a), cph1 (b), efg1 (c), cph1/efg1 (d) and the EFG1 revertant (e) after incubation with antibodies directed against Sap1–3. There was strong gold labelling within the cell wall of SC5314 (a), cph1 (b) and the EFG1 revertant (e) cells. efg1 (c) and the cph1/efg1 (d) mutant cells are labelled with only a few gold particles.

Cytokine response is modulated only by the cph1/efg1 double mutant

We investigated the C. albicans-induced expression of IL-1ß, IL-8 and TNF of the RHE because a chemotactic signal for the recruitment of PMNs (IL-1ß and IL-8) and a Th-1 response (TNF) is crucial for stimulating a protective immune response to the site of mucosal infection.

Non-infected RHE showed expression of genes encoding aldolase and GAPDH but no IL-1ß, IL-8 and TNF transcripts. The wild-type and the cph1 and the efg1 mutants demonstrated a similar ability to stimulate epithelial expression of IL-1ß, IL-8 and TNF 12, 36, 46 and 52 h after infection (Fig. 4). In contrast, the cph1/efg1 mutant failed to stimulate epithelial IL-1ß and IL-8 expression at 12 and 36 h, and TNF expression at 12, 36 and 46 h after inoculation of RHE (Fig. 4).

View larger version (69K): [in this window] [in a new window]   Fig. 4. Kinetics of cytokine expression (IL-1ß, IL-8 and TNF) by RHE 12, 36, 46 and 52 h after stimulation with SC5314 (wild-type), cph1, efg1 and cph1/efg1. Aldolase was used as an internal mRNA control. Results were obtained by RT-PCR. Failure of epithelial IL-1ß and IL-8 expression at 12 and 36 h and TNF expression at 12, 36 and 46 h after infection of RHE with the cph1/efg1 mutant as compared to cytokine expression after wild-type, cph1 and efg1 infection is shown.

	DISCUSSION

TOP Abstract INTRODUCTION METHODS RESULTS DISCUSSION Acknowledgements REFERENCES

 
C. albicans possesses a variety of virulence attributes such as dimorphism, proteolytic enzymes and phenotypic switching necessary for tissue invasion (Calderone & Fonzi, 2001). In recent years it has been proposed that these single virulence factors may act synergistically to improve the virulence properties of the pathogen (Sonneborn et al., 1999b; Schröppel et al., 2000; Staib et al., 2002; Felk et al., 2002; Schweizer et al., 2000; Srikantha et al., 2000). Results from these experiments showed that transcription factors such as Efg1 or CaTec1 influenced the regulation of certain virulence attributes, for example phenotypic switching (Sonneborn et al., 1999b; Srikantha et al., 2000) or the expression of secreted aspartyl proteinases (Schröppel et al., 2000; Staib et al., 2002; Felk et al., 2002; Schweizer et al., 2000). Investigations of a correlation between dimorphism and expression of proteinase genes in these studies concentrated on the hyphal-associated genes SAP4–SAP6 (Schröppel et al., 2000; Staib et al., 2002; Felk et al., 2002; Schweizer et al., 2000). Recent gene array data have revealed that thousands of C. albicans genes are regulated during the yeast-to-hyphal transition, suggesting that a network of genes is involved during these morphological changes (Lane et al., 2001). Our data demonstrate that the disruption of transcription factors which regulate the yeast-to-hyphal transition also has a consequence on the expression of hyphal-independent genes such as the proteinase genes SAP1 and SAP3.

The majority of studies dealing with the virulence potential of EFG1- and/or CPH1-deficient mutants used murine or murine cell type models. Differences in the virulence of cph1, efg1 single and the cph1/efg1 double mutants were found in a mouse model for systemic infection (Lo et al., 1997; Staib et al., 2002; Felk et al., 2002) and during interaction with mouse macrophages (Lo et al., 1997). Investigations using human tissues were performed with endothelial (Phan et al., 2000), intestinal and epidermal cells (Dieterich et al., 2002). Results from these experiments demonstrated a significantly decreased virulence phenotype only in the mutants lacking functional EFG1 but not for the cph1 single mutant. In this study, we present evidence for an important role of dimorphism and hyphal-associated factors during invasion of human oral epithelium and during interaction with human neutrophils. These are novel contributions to the understanding of host–pathogen interactions in oral candidosis as oral epithelium is different from endothelial, epidermal or intestinal cells concerning morphology, function and expression of cytokines and markers of differentiation (Li et al., 2000; Li & Thornhill, 2000; Uehara et al., 2001). The interaction of murine macrophages with CPH1- and/or EFG1-deficient C. albicans mutants has been published by Lo et al. (1997). PMNs, however, are microphages and belong to the first line of host defence in oral candidosis (Eversole et al., 1997; Challacombe, 1994; Farah et al., 2001), while macrophages seem to play a more important role in deep-seated or systemic infections (Vazquez-Torres & Balish, 1997). Therefore, we decided to investigate the virulence phenotype of these mutants also during interaction with PMNs as an important host–fungus interaction in oral candidosis. Our results indicate that the Efg1-regulated PKA signal transduction pathway, but not the Cph1-regulated MAPK pathway, is important for the ability of C. albicans to damage human oral keratinocytes and to survive phagocytosis by human PMNs.

In previous studies, we have shown that SAP1, SAP2 and SAP3 are crucial for epithelial damage in a model of oral candidosis based on RHE (Schaller et al., 1998, 1999). Therefore, we focused our studies on the expression of these SAP genes, which are especially important for this type of infection. In addition to morphological alterations, another possible reason for the attenuated pathogenicity of the hyphal-deficient efg1 and cph1/efg1 strains during infection of human oral epithelium might be a reduced level of SAP expression. During experimental oral infection, both virulence-attenuated mutants correspondingly failed to express detectable amounts of SAP1 and SAP3 transcripts as compared with the parental wild-type, the cph1 mutant and the EFG1 revertant strains. Decreased secretion of Sap1–3 by efg1 and cph1/efg1 was also confirmed on the protein level by immunoelectron microscopical studies. As shown in this study, deletion of CPH1 did not reduce the ability to injure human keratinocytes and PMNs or change the SAP expression pattern during infection of human oral epithelium as compared with the parental strain. Modulation of SAP expression by EFG1 was previously investigated by Schröppel et al. (2000) using Northern analysis. They demonstrated that efg1 failed to express SAP4, SAP5 and SAP6 up to 4 h after induction under distinct in vitro hyphal-inducing conditions. In our hands, using RT-PCR, expression of SAP5 and SAP6 by this mutant but not by cph1/efg1 was detected during interaction with keratinocytes. A similar modulation of SAP5 in the same mutants during systemic disease was found by Staib et al. (2002), who used an in vivo expression technology. In their study, transcript levels were reduced but not eliminated in efg1 and completely lost in cph1/efg1 during systemic mouse infection (Staib et al., 2002). Felk et al. (2002) demonstrated strongly reduced transcript levels of SAP4, SAP5 and SAP6 by RT-PCR under certain, but not all, in vitro hyphal induction conditions and during in vivo expression in the peritoneal cavity of infected mice. These observed differences in these studies might be due to the various environmental conditions during the different growth conditions or types of infection, which may differentially modulate gene expression. Another possible reason might be the different sensitivity of the detection techniques used in each study. In addition to the well-known regulation of SAP4, SAP5 and SAP6 by EFG1 we were able to demonstrate that expression of SAP1 and SAP3 is also modulated in mutants lacking this factor. It remains to be tested whether this is a direct consequence of a lack of transcriptional activation or an indirect effect, for example due to altered growth or virulence properties of these mutants. Amplification of the cDNA with primers for the house-keeping gene EFB1 of C. albicans demonstrated similar expression levels for all investigated strains. Therefore, reduced cell growth of the mutants seems to be an unlikely explanation for the observed phenotypes.

Stimulation of cytokine and chemokine expression during infection of human oral epithelium was identical for the parental strain and the single mutants, while immune response was reduced for the cph1/efg1 infection, suggesting a synergistic effect of the simultaneous deletion of CPH1 and EFG1. Since the hyphal-deficient mutant efg1 caused a similar cytokine expression pattern to wild-type cells, we concluded that hyphal morphology does not contribute significantly to the observed cytokine pattern. A similar pattern for expression of leukocyte adhesion molecules by endothelial cells induced by these different strains of C. albicans was demonstrated recently (Phan et al., 2000).

Using a new set of highly specific SAP1–10 primers, we were able to confirm our previous studies (Schaller et al., 1998, 1999). We found a differential SAP gene expression pattern during experimental epithelial infection and could confirm that the presence of SAP1 and SAP3 transcripts correlates with the onset of epithelial lesions. Expression of SAP2, SAP5 and SAP6 was also seen in earlier stages without causing significant histological damage, indicating that these genes are not important for the development of a manifest oral disease as previously postulated by Naglik et al. (1999). In former studies, expression of SAP9 and SAP10 was not investigated. Here we show that both genes were expressed during all chosen growth conditions of the wild-type and the mutant strains, suggesting that the expression of these genes is independent from Cph1 and Efg1.

In summary, we present evidence that Efg1 is a key regulator for virulence during interaction with human epithelial cells and PMNs not only by regulating dimorphism but also by influencing the expression of hyphal-dependent and hyphal-independent aspartyl proteinase genes during experimental oral infection.

	Acknowledgements

TOP Abstract INTRODUCTION METHODS RESULTS DISCUSSION Acknowledgements REFERENCES

 
We thank J. Ernst, Department of Microbiology, Heinrich–Heine-University of Düsseldorf, Düsseldorf, Germany, and G. Fink, Whitehead Insitute for Biomedical Research, Cambridge, MA, for providing the cph1, efg1 and cph1/efg1 mutants and the EFG1 revertant and W. Burgdorf for critical reading of the manuscript.

This study was supported by the Deutsche Forschungsgemeinschaft for M. S. (KO 1106/4-1, SCH 897/1-2), H. C. K. (KO 1106/4-1) and B. H. (Hu 528/7 and Hu 528/8) and the European Commission for B. H. (QLK2-2000-00795).

	REFERENCES

TOP Abstract INTRODUCTION METHODS RESULTS DISCUSSION Acknowledgements REFERENCES